Modular protein libraries and methods of preparation

ABSTRACT

Novel proteins and libraries comprising them are disclosed. The proteins comprise one or more functional protein modules from different parent protein molecules. The proteins and libraries are exemplified by the preparation of cross-over chemokines comprising various combinations of peptide segments derived from RANTES, SDF-1 and vMIP-I and vMIP-II. The proteins and libraries are extremely pure and can be provided in non-limiting high yields suitable for diagnostic and high-throughput screening assays.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to provisional application U.S.Serial No. 60/057,620, filed Sep. 4, 1997.

FIELD OF THE INVENTION

[0002] The present invention relates to modular protein molecules andmodular protein libraries obtained by cross-over synthesis of two ormore functional protein modules derived from different parent proteinmolecules.

BACKGROUND OF THE INVENTION

[0003] Chemical leads for the pharmaceutical industry are currentlyidentified through rational design and/or mass screening. The recentintroduction of high throughput, automated screening technologies haspermitted evaluation of hundreds of thousands of individual testmolecules against a large number of targets. However, the source,diversity and functionality of large chemical libraries still remains alimitation in identifying new leads. Compound libraries commonly used inmass screening consist of either a historical collection of synthesizedcompounds or natural product collections. Historical collections containa limited number of diverse structures and represent only a smallfraction of diversity possibilities. They also contain a limited numberof biologically useful compounds. Natural products are limited by thestructural complexity of the leads identified and the difficulty ofreducing them to useful pharmaceutical agents (e.g., taxol).

[0004] Methods available for generating synthetic compound librariesdiffer considerably in the types and numbers of compounds prepared, andwhether the compounds are obtained as single structurally definedentities or as large mixtures. New compound libraries have been obtainedthrough rapid chemical and biological synthesis (Moos et al., Ann. Rep.Med. Chem. (1993) 28:315-324; Pavia et al., Bioorganic Medicinal Chem.Lett. (1993) 3:387-96; Gallop et al., J. Med. Chem., (1994)37:1233-1251; Gordon et al, J. Med. Chem. (1994) 37:1385-1401). Peptidelibraries containing hundreds to millions of small to medium sizepeptides have been made using “pin technology” representing a methodthat generates libraries of single compounds in aspatially-differentiated manner (Geysen et al., Proc. Nat. Acad. Sci.U.S.A. (1984), 81:3998-4002). The “spilt pool” method provides analternative approach to preparing large mixtures of peptides and otherclasses of molecules (Furka et al., Abstr. 14th Int. Congr. Biochem.,Prague, Czechoslovakia, Vol 5, pg 47. Abstr. 10th Intl. Symp. Med.Chem., Budapest, Hungary, (1988), pg 288; Houghten et al., Proc. Natl.Acad. Sci. U.S.A. (1985) 82:5131-35). Peptide libraries also have beenproduced by the “tea-bag” method in which small amounts of resinsrepresenting individual peptides are enclosed in porous polypropylenecontainers (Houghten et al., Nature (1991) 354:84-86). The bags areimmersed in individual solutions of the appropriate activated aminoacids while deprotections and washings are carried out by mixing all thebags together. The bags are then reseparated for subsequent couplingsteps (the split-pool method). Removal of the peptides from the resinsaffords peptides in soluble form. It is possible to rapidly prepare acollection of libraries which represents, for example, all 64 millionnaturally-occurring hexapeptides and identify an optimal peptide ligandfor any ligate of interest. Libraries of peptides also have beenprepared on polymeric beads by the split-pool method and incubated witha tagged ligate. Ligates with bound peptides are identified by visualinspection, physically removed, and microsequenced (Lam et al., Nature(1991) 354:82-84). The approach also can incorporate cleavable linkerson each bead where, after exposure to cleaving reagent, the beadsrelease a portion of their peptides into solution for biological assayand still retain sufficient peptide on the bead for microsequencing. Thepin, split-pool, and tea-bag methods and libraries generated therefromare limited to relatively small peptides amenable to this technology andthe difficulty in identifying functional peptides of interest.

[0005] Peptide libraries also have been prepared in which an“identifier” tag is attached to a solid support material coincident witheach monomer using a split-pool synthesis procedure. The structure ofthe molecule on any bead identified through screening is obtained bydecoding the identifier tags. Numerous methods of tagging the beads havenow been reported. These include the use of single strandedoligonucleotides which have the advantage of being used as identifyingtags as well as allowing for enrichment through the use for PCRamplification (Brenner et al., Proc. Natl. Acad. Sci. U.S.A. (1992)89:5381-5383; Nielsen et al., J. Am. Chem. Soc. (1993) 115:9812-9813;Needels et al., Proc. Natl. Acad. Sci. USA (1993) 90:10700-10704). Theuse of halocarbon derivatives which are released from the active beadsthrough photolysis and sequenced using electron capture capillarychromatography has also been described (Gallop et al., Journal ofMedicinal Chemistry, (1994) 37:1233-1251). While identifier tags aidscreening of large peptide libraries, peptides are likely to havelimited therapeutic applicability when modulation of receptor activityinvolved in a particular disorder require interaction with wholeproteins, or protein complexes.

[0006] Phage libraries containing tens of millions of filamentous phageclones have been used as a biological source for generating peptidelibraries, with each clone displaying a unique peptide sequence on thebacteriophage surface (Smith G. P., Science (1985) 228:1315-1317; Cwirlaet al., Proc. Natl. Acad. Sci. USA (1990) 87:6378-6382; Devlin et al.,Science (1990) 249:404-406). In this method, the phage genome containsthe DNA sequence encoding for the peptide. The ligate of interest isused to affinity purify phage that display binding peptides, the phagepropagated in E. coli, and the amino acid sequences of the peptidesdisplayed on the phage are identified by sequencing the correspondingcoding region of the viral DNA. Tens of millions of peptides can berapidly surveyed for binding. Initial libraries of short peptidesgenerally afford relatively weak ligands. Longer epitope regions and/orconstrained epitopes also have been prepared. Phage technology also haseffectively been applied to proteins and antibodies demonstrating thatprotein domains can fold properly on the surface of phage. A limitationof this method is that only naturally occurring amino acids can be usedand little is known about the effect of the phage environment, as wellas contaminants from cellular debris and phage.

[0007] Peptoid libraries have been created which represent a collectionof peptides having N-substituted glycines as peptoid monomers(Zuckermann et al., J. Med. Chem. (1994) 37: 2678-2685; Bunin et al., J.Am. Chem. Soc. (1992) 114:10997-10998; DeWitt et al., Proc. Natl. Acad.Sci. U.S.A. (1993) 90:6909-6913; Bunin et al., Proc. Natl. Acad. Sci.USA (1993) 91:4708-4712; Hogan et al. WO 94/01102). Structures of theresulting compounds are unique, likely to display unique bindingproperties, and incorporate important functionalities of peptides in anovel backbone. The methods generate single structurally well definedmolecules in a solution format after cleavage from a solid support. Adisadvantage of this approach is the lack of correlating structure withfunction in screening the modified peptides, as well as limitedtherapeutic application when small peptides are insufficient to mimicactivity of a protein or protein complex.

[0008] While each of the technologies described above afford a largenumber of compounds, the usefulness of these systems for the effectiverapid discovery of drug candidates is limited since all of them resultin the identification of relatively small peptide ligands. In mostcases, small peptides are not suited as drugs due to in vivo instabilityand lack of oral absorption. Furthermore, conversion of a peptidechemical lead into a pharmaceutically useful, orally active, non-peptidedrug candidate is more difficult than identifying the original peptidelead since no general solution yet exists for designing effectivepeptide mimics.

[0009] Another significant limitation of the various approachesdescribed above are the size and complexity of the libraries, whetherthey are generated as single compounds (active compound identified byit's physical location) or mixtures (active compound identified by it'stag for encoded libraries or through deconvolution, where an activecompound is identified by iterative synthesis and screening ofmixtures). In addition, the construction of random synthetic, native,and phage libraries have proven useful but fall short of providing amore rational approach in development of compound libraries for theidentification of a novel lead chemical structure. Accordingly, thereexists a need to develop new libraries comprising functionally diversecompounds to improve the drug discovery process.

RELEVANT LITERATURE

[0010] Peptide libraries constructed by chemical synthesis have beendisclosed by Hogan et al., (WO 94/01102). Dawson et al. (Science (1994)266:776-779) and Kent et al. (WO 96/34878) disclose a method for thechemical synthesis of proteins by native chemical ligation. Variouscombinations of solid and solution phase ligation technologies for thesynthesis of chemokines and analogues also have been disclosed (Siani etal., IBC 3rd Annual International Conference: Chemokines, September1996; Siani et al., NMHCC, Chemokines and Host-Cell InteractionConference, January 1997, Baltimore, Maryland; Siani et al., PeptideSymposium, Nashville, June 1997; Canne et al., American PeptideSymposium, Nashville, June 1997; and Siani, et al., American Peptide,Jun. 15-19, 1997, Nashville, Tenn.). Wernette-Hammond et al. (J. Biol.Chem. (1996) 271:8228-8235) disclose recombinant expression of chimericproteins comprising segments from IL-8 and GRO-gamma.

SUMMARY OF THE INVENTION

[0011] Novel proteins comprising a combination of two or more functionalmodules from two or more different parent proteins, and librariescomprising the proteins are provided. The proteins and libraries of theinvention are produced by cross-over synthesis of functional proteinmodules identified among a class or family of proteins. Librariescomprising novel cross-over chemokines are exemplified. The presentinvention includes novel therapeutic leads and compounds forcharacterizing the chemical basis of known ligand/ligate interactionsincluding epitope mapping, receptor localization and isolation. Themethods of the invention are applicable to other families of proteins inaddition to the chemokines for diversity generation of libraries andpharmaceutical leads.

[0012] The cross-over protein libraries of the invention permitrefinement of specific properties of particular protein molecules,including activity, stability, specificity and immunogenicity. Theprocess begins with the generation of a focused set of candidate proteinanalogues based on a protein family identified as having functionalmodules. The functional protein modules can be identified by any numberof means including identification of structure and functionrelationships. Structural relationships are preferably based on homologycomparisons between nucleotide, amino acid, and/or three-dimensionalanalysis. The structural components can be assessed separately or incombination with functional analysis including assays which correlatestructural data with a particular activity. The cross-over proteins ofthe invention are then prepared by ligation of the functional modules toform a single polypeptide chain. A preferred method of modular proteinsynthesis employs chemical ligation to join together large peptidesegments to form functional polypeptides or proteins. A combination ofpeptide synthesis and one or more ligation steps also can be used. Solidphase and native chemical ligation techniques are preferred forconstructing the cross-over proteins.

[0013] The modular protein synthesis approach permits an efficient andhigh-yield method for the construction of synthetic protein libraries ofhybrid molecules that can be much larger than is possible withconventional synthesis techniques. After functional selection, proteinmolecules with desired characteristics are identified and then used asleads for subsequent cycles of synthesis and screening. The speed ofmodular chemical synthesis and the efficiency of the analogueidentification methods enable multiple rounds of refinement to producefinely-tuned protein therapeutic candidates. Additionally, chemicalligation permits unprecedented access to extremely pure cross-overprotein libraries free of cellular contaminants.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 shows a general method for generating molecular diversityby cross-over synthesis of CXC and CC chemokines.

[0015]FIG. 2 shows a method for generating molecular diversity bycross-over synthesis of the CXC chemokine SDF-1α and the CC chemokineRANTES.

[0016]FIG. 3 shows chemokine amino acid sequence patterns for RANTES,SDF-1α and MPBV.

[0017]FIG. 4 shows analytical HPLC chromatograms for SSSS (control) andS′SSS, SRRR, and S′RRR modular chemokines; conditions: C4 reversed-phaseHPLC column running a gradient of 5%-65% acetonitrile versus watercontaining 0.1% TFA, over 30 minutes, with detection at 214 nm.

[0018]FIG. 5 shows analytical HPLC chromatograms for RRRR (control) andR′RRR, RSSS, and R′SSS modular chemokines; conditions: C4 reversed-phaseHPLC column running a gradient of 5%-65% acetonitrile versus watercontaining 0.1% TFA, over 30 minutes, with detection at 214 mm.

DEFINITIONS

[0019] “Peptide.” Two or more amino acids operatively joined by apeptide bond. By operatively joined it is intended that the structureand function of a peptide bond in a naturally occurring protein isrepresented.

[0020] “Protein.” Two or more peptides operatively joined by a peptidebond. The term protein is interchangeable with the term polypeptide.

[0021] “Functional Protein Module.” A segment of a protein comprising asequence of amino acids that provides a particular functionality in afolded protein. The functionality is based on positioning of thesequence in three-dimensional space and can be formed by two or morediscontinuous protein sequences.

[0022] “Modular Protein.” A protein comprising a combination of two ormore functional protein modules operatively joined by one or morepeptide bonds.

[0023] “Modular Protein Library.” A collection of modular proteincompounds.

[0024] “Cross-Over Protein.” A hybrid protein comprising one or morefunctional protein modules derived from different parent proteinmolecules. The functional protein modules are provided by two or morepeptide segments joined by a native or non-native peptide bond. Thesegments can comprise native amide bonds or any of the known unnaturalpeptide backbones or a mixture thereof. May include the 20 geneticallycoded amino acids, rare or unusual amino acids that are found in nature,and any of the non-naturally occurring and modified amino acids.

[0025] “Cross-Over Protein Library.” A collection of cross-over proteincompounds.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides cross-over proteins produced bychemical ligation of two or more functional protein modules derived fromtwo or more different parent protein molecules. The chemical ligationinvolves ligating under chemoselective chemical ligation conditions atleast one N-terminal peptide segment comprising a functional proteinmodule of a first parent protein and at least one C-terminal peptidesegment comprising a functional protein module of a second parentprotein, where the N-terminal and C-terminal peptide segments providecompatible reactive groups capable of chemoselective chemical ligation.The first and second parent proteins preferably are members of the samefamily of proteins, and may include one or more mutations relative to anaturally occurring parent protein molecule.

[0027] The cross-over proteins and methods of the invention provideunprecedented access to new proteins molecules useful for multiplediagnostic and drug discovery applications. For example, proteins act onreceptors to elicit a characteristic biological response. Proteins arecomposed of functional modules that have functionality relative to thefolded protein. Accordingly, cross-over ligation of two or moredifferent functional modules from different proteins of a class orfamily generates new hybrid protein molecules. The cross-over proteinsof the invention have unique properties that can be used to evaluatefunction and tune desired properties, such as biological activity aswell as physicochemical properties related to formulation andadministration.

[0028] The cross-over proteins of the invention also may include one ormore modified amino acids, such as an amino acid comprising a chemicaltag. The chemical tag may be introduced during and/or after synthesis ofthe cross-over protein molecule. The chemical tag may be utilized formultiple purposes such as part of the synthesis process, purification,anchoring to a support matrix, detection and the like. Of particularinterest is a chemical tag provided by an unnatural amino acidcomprising a chromophore. This includes a chromophore that is anacceptor and/or donor moiety of an acceptor-donor resonance energytransfer pair.

[0029] The present invention also provides libraries of cross-overproteins. A collection of cross-over proteins derived from a particularclass or family of protein molecules represents a focused and rationallydesigned library of novel and structurally diverse cross-over proteinmolecules that permit collective analysis and identification oftherapeutic leads that can combine properties contributed by two or moredistinct parent proteins. A preferred cross-over protein library of theinvention contains at least four or more unique cross-over proteins.

[0030] Libraries of cross-over proteins of the invention are prepared byligation of distinct functional modules from a particular class orfamily of proteins. The functional modules may be identified bycomparing nucleotide and/or amino acid sequence information of a targetprotein to identify one or more modules representing a particularfunctionality for the protein family. Computer analysis, simulation andatomic coordinate information also may be employed for comparison. Asbiological macromolecules (receptor, enzyme, antibody, etc.) recognizebinding substrates through a number of precise physicochemicalinteractions, these interactions can be divided into a number ofdifferent parameters or dimensions such as size, hydrogen bondingability, hydrophobic interactions, etc., each of which contribute to theactivity of a functional protein module. Functional modules fromdifferent proteins having distinct biological activity within the familyare selected to maintain the basic three-dimensional scaffold of theinitial class of target molecule. The cross-over protein libraries aretherefore designed to orient groups responsible for binding interactionsat unique locations in three-dimensional space relative to a rudimentaryprotein scaffold. This allows for facile introduction of two or morefunctional groups in a large number of spatial arrangements. A largenumber of compounds prepared around each scaffold will reflect a diverserange of unique activities, sizes, shapes, and volumes.

[0031] Additional diversity can be added to the library throughsubsequent chemical modification of the proteins, such as amino and/orcarboxyl terminal modification, and/or the incorporation of non-naturalamino acids. Another example includes synthesis of functional modules ofdefined structure and length, where specified positions or a definednumber of positions contain a random mixture of amino acids.

[0032] A double combinatorial approach also can be used in whichfunctional groups, representing various physicochemical interactingproperties, are introduced by combining functional protein modules intothe scaffold building block. A second scaffold building block can beadded followed by an additional round of functional group introduction.The final target molecule is prepared for screening. This approachpermits the rapid production of a second or sub-library of highlyfunctionalized target molecules from the first library, which mayrepresent only a small collection of functional protein modules.

[0033] The cross-over proteins of the invention are generated bychemical ligation techniques. The chemical ligation method of theinvention involves cross-over chemoselective chemical ligation of (i) atleast one functional N-terminal peptide segment comprising one or morefunctional protein modules derived from a first parent protein, and (ii)at least one functional C-terminal peptide segment comprising one ormore functional protein modules derived from a second parent proteinhaving one or more properties and an amino acid sequence that isdifferent from the first parent protein under chemoselective chemicalligation conditions, where the N-terminal peptide segment and theC-terminal peptide segment provide compatible reactive groups capable ofchemoselective chemical ligation. The cross-over ligation reaction isallowed to proceed under conditions whereby a covalent bond is formedbetween the N-terminal and C-terminal peptide segments so as to producea chemical ligation product comprising a cross-over protein.

[0034] A peptide segment utilized for construction of a cross-overprotein of the invention contains an N-terminus and a C-terminus withrespect to directionality of the amino acid sequence comprising thesegment. For a given chemical ligation event, two protein segments, eachcomprising one or more functional protein modules, form a covalent bondbetween a reactive group donated by an amino acid of the N-terminal endof the first segment and a reactive group donated by an amino acid ofthe C-terminal end of the second segment (i.e., head to tail chemicalligation). Thus use of the terminology “N-terminal peptide segment” and“C-terminal peptide segment” refers to the directionality of the proteinsegment relative to a particular chemoselective ligation event and/orthe final cross-over protein product. By way of example, and withreference to FIGS. 1-2 illustrating cross-over ligation of the CXC andCC chemokines SDF1α (S) and RANTES (R), respectively, a given cross-overchemokine exemplified in FIGS. 1-2 may be formed by chemical ligationutilizing protein or peptide segments that comprise one or morefunctional modules (S, S′, R and R′), such as two peptide segments(e.g., ligation of SS′ (N-terminal) and RR′ (C-terminal) to yield SS′RR′cross-over chemokine), three peptide segments (e.g., ligation of S(N-terminal) and S′R (C-terminal) to yield SS′R (N-terminal), followedby ligation of SS′R (N-terminal) to R′ (C-terminal) to yield SS′RR′cross-over protein), or four peptide segments (e.g., ligation of S(N-terminal) and S′ (C-terminal) to yield SS′ (N-terminal), and ligationof R (N-terminal) and R′ (C-terminal) to yield RR′ (C-terminal),followed by ligation of SS′ (N-terminal) and RR′ (C-terminal) to yieldSS′RR′ cross-over chemokine). As can be appreciated, any number ofmodular combinations and ligation orders are possible.

[0035] The cross-over ligations may be performed in single or separatereactions, and optionally include a plurality of chemoselective ligationcompatible N- and C-terminal peptide segments representing a mixture offunctional protein modules derived from two or more different parentproteins, so as to obtain a plurality of unique cross-over proteins.When a mixture of unique N-terminal and C-terminal peptide segments areemployed, the ligated products can be identified and separated fromnon-specific side reactions and unligated components by any number ofseparation techniques, such affinity or high performance liquidchromatography. Further deconvolution can be utilized to pool orseparate the desired ligation products. Also, the mixtures may representspecific groups or sub-groups of peptide segments so as to regulate thenumber of possible desired ligation outcomes per reaction. One or moreinternal controls (e.g., parent protein molecules), or coding tags(e.g., chemically tagged cross-over ligation peptide segments) may beincluded to ease deconvolution. Activity screens also may be used inconjunction with deconvolution.

[0036] In a preferred embodiment, one or more of the N-terminal andC-terminal peptide segments utilized in a given cross-over chemicalligation are pre-formed by cross-over ligation, which are then employedfor construction of cross-over proteins. This aspect of the inventioninvolves cross-over ligation of two or more functional protein modulesderived from different parent proteins of the same family by (i)generating a plurality of functional N-terminal peptide segments havingone or more functional protein modules obtained by cross-over ligationof two or more different parent protein molecules, and a plurality offunctional C-terminal peptide segments having one or more functionalprotein modules obtained by cross-over ligation of two or more differentparent protein molecules, followed by (ii) cross-over ligation of theplurality of cross-over N-terminal and C-terminal modules so as toobtain a plurality of unique cross-over proteins.

[0037] One of ordinary skill in the art will recognize that the largerthe library of unique cross-over proteins, the greater the diversity andinformation and leads derivable therefrom. The size and diversity of alibrary can be determined by calculating the number of possible uniquecross-over events based on the number of unique N-terminal andC-terminal modules as described above. This may employ simulations,modeling and the like as a basis for designing cross-over proteins ofthe invention, followed by synthesis and screening of the cross-overmolecules for activity. It also will be appreciated by one of ordinaryskill that molecules exhibiting activity, a range of activity or noactivity for a given screening assay provide useful structure-activityrelationship (SAR) and quantitative SAR (QSAR) information forcharacterizing structure-function of individual modules and combinationsof modules, and thus iterative design, screening and synthesis. Forinstance, libraries can be generated by computer simulation (virtuallibrary) followed by synthesis employing the combinatorial ligationchemistry approaches of the invention (physical library). The physicallibraries then can be screened in a biological assay and resultingactivity profiles assessed relative to a given functionality imparted,modified or otherwise removed and the like by a module or combination ofmodules.

[0038] The cross-over proteins can be made to resemble or duplicatefeatures of naturally occurring peptides or segments of naturallyoccurring proteins. The design of a particular cross-over protein isbased on its intended use and on considerations of the method ofsynthesis. As the proteins increase in length, they have a greatertendency to adopt elements of secondary structure such as loops,α-helicies and β-sheet structures connected by discrete turns, whichimpart an overall decrease in flexibility. These elements in part arethe components which comprise a scaffold that present functional groupsresponsible for specific biological activity. From knowledge of thefeatures that contribute to these structures, the proteins can bespecifically designed to contain them. Of particular interest arecross-over protein molecules synthesized by combining a functionalmodule from a first protein with a functional module from a secondprotein. Additional functional modules can be combined from the sameand/or one or more other proteins. A preferred cross-over protein isproduced by combining one or more functional modules from a firstchemokine and a second chemokine. The cross-over protein molecules areassayed for biological activity, for example, the cross-over chemokinesare evaluated for induction of lyphocyte chemotaxis and binding toreceptors.

[0039] The cross-over proteins can be linear, cyclic or branched, andoften composed of, but not limited to, the 20 genetically encodedL-amino acids. A chemical synthetic approach permits incorporation ofnovel or unusual chemical moieties including D-amino acids, otherunnatural amino acids, ester or alkyl backbone bonds in place of thenormal amide bond, N- or C-alkyl subtituents, side chain modifications,and constraints such as disulfide bridges and side chain amide or esterlinkages. The chemical modification is designed to impart changes inbiological potency, stability related to halflife in vivo and storage,and the ability to interact with or covalently label a biologicalmacromolecule receptor for localization of structure-function assays.

[0040] Peptide segments utilized for initial ligation and synthesis ofthe cross-over proteins of the invention may be synthesized chemically,ribosomally in a cell free system, ribosomally within a cell, or anycombination thereof. Accordingly, cross-over proteins generated byligation according to the method of the invention include totallysynthetic and semi-synthetic cross-over proteins. Ribosomal synthesismay employ any number of recombinant DNA and expression techniques,which techniques are well known. See, for example, Sambrook et al.(1989, “Molecular Cloning, A Laboratory Manual,” Cold Springs HarborPress, New York); “Recombinant Gene Expression Protocols,” Humana Press,1996; and Ausubel et al. (1989, “Current Protocols in MolecularBiology,” Green Publishing Associates and Wiley Interscience, New York).For chemical synthesis, peptide segments can be synthesized either insolution, solid phase or a combination of these methods followingstandard protocols. See, for example, Wilken et al. (Curr. Opin.Biotech. (1998) 9(4):412-426), which reviews chemical protein synthesistechniques. The solution and solid phase synthesis methods are readilyautomated. A variety of peptide synthesizers are commercially availablefor batchwise and continuous flow operations as well as for thesynthesis of multiple peptides within the same run. The solid phasemethod consists basically of anchoring the growing peptide chain to aninsoluble support or resin. This is accomplished through the use of achemical handle, which links the support to the first amino acid at thecarboxyl terminus of the peptide. Subsequent amino acids are then addedin a stepwise fashion one at a time until the peptide segment is fullyconstructed. Solid phase chemistry has the advantage of permittingremoval of excess reagents and soluble reaction by products byfiltration and washing. The protecting groups of the fully assembledresin bound peptide chain are removed by standard chemistries suitablefor this purpose. Standard chemistries also may be employed to removethe peptide chain from the resin. Cleavable linkers can be employed forthis purpose. For solution phase peptide synthesis this generallyinvolves reacting individual protected amino acids in solution togenerate protected dipeptide product. After removal of a protectiongroup to expose a reactive group for addition of the next amino acid, asecond protected amino acid is reacted to this group to give a protectedtripeptide. The process of deprotection/amino acid addition is repeatedin a stepwise fashion to yield a protected peptide product. One or moreto these protected peptides can be reacted to give the full-lengthprotected peptide. Most or all or the remaining protecting groups areremoved to generate an unprotected synthetic peptide segment. Thus,solid phase or solution phase chemistries may be employed to formsynthetic peptides comprising one or more functional protein modules.

[0041] The preferred method of synthesis employs a combination ofchemical synthesis and chemical ligation techniques. By way of example,chemical synthesis approaches described above may be utilized incombination with various chemoselective chemical ligation techniques forproducing the cross-over proteins of the invention. Chemoselectivechemical ligation chemistries that can be utilized in the methods of theinvention include native chemical ligation (Dawson et al., Science(1994) 266:77-779; Kent et al., WO 96/34878), extended general chemicalligation (Kent et al., WO 98/28434), oxime-forming chemical ligation(Rose et al., J. Amer. Chem. Soc. (1994) 116:30-33), thioester formingligation (Schnolzer et al., Science (1992) 256:221-225), thioetherforming ligation (Englebretsen et al., Tet. Letts. (1995)36(48):8871-8874), hydrazone forming ligation (Gaertner et al., Bioconj.Chem. (1994) 5(4):333-338). thaizolidine forming ligation andoxazolidine forming ligation (Zhang et al., Proc. Natl. Acad. Sci.(1998) 95(16):9184-9189; Tam et al., WO 95/00846). The preferredchemical ligation chemistry for synthesis of cross-over proteinsaccording to the method of the invention is native chemical ligation.

[0042] For example, the synthesis of proteins by native chemicalligation is disclosed in Kent et al., WO 96/34878. In general, a firstoligopeptide containing a C-terminal thioester is reacted with a secondoligopeptide with an N-terminal cysteine having an unoxidized sulfhydrylside chain. The unoxidized sulfhydryl side chain of the N-terminalcysteine is condensed with the C-terminal thioester in the presence of acatalytic amount of a thiol, preferably benzyl mercaptan, thiophenol,2-nitrothiophenol, 2-thiobenzoic acid, 2-thiopyridine, and the like. Anintermediate oligopeptide is produced by linking the first and secondoligopeptides via a β-aminothioester bond, which rearranges to producean oligopeptide product comprising the first and second oligopeptideslinked by an amide bond.

[0043] Synthesis of cross-over proteins according to the methods of theinvention by a combination of chemical ligation and chemical synthesispermits facile incorporation of one or more chemical tags. These includesynthesis and purification handles, as well as detectable labels andoptionally chemical moieties for attaching the cross-over protein to asupport matrix for screening and diagnostic assays and the like. As canbe appreciated, in some instances it may be advantageous to utilize agiven chemical tag for more than one purpose, e.g., both as a handle forattaching to support matrix and as a detectable label. Examples ofchemical tags include metal binding tags (e.g., his-tags),carbohydrate/substrate binding tags (e.g., cellulose and chitin bindingdomains), antibodies and antibody fragment tags, isotopic labels,haptens such as biotin and various unnatural amino acids comprising achromophore. A chemical tag also may include a cleavable linker so as topermit separation of the cross-over protein from the chemical tagdepending on its intended end use.

[0044] For example, it may be convenient to conjugate a fluorophore tothe N-terminus of a resin-bound peptide utilized for synthesis andligation of cross-over proteins of the invention before removal of otherprotecting groups and release of the labeled peptide from the resin.About five equivalents of an amine-reactive fluorophore are usually usedper amine of the immobilized peptide. Fluorescein, eosin, Oregon Green,Rhodamine Green, Rhodol Green, tetramethylrhodamine, Rhodamine Red,Texas Red, coumarin and NBD fluorophores, the dabcyl chromophore andbiotin are all reasonably stable to hydrogen fluoride (HF), as well asto most other acids. (Peled et al., Biochemistry (1994) 33:7211;Ben-Efraim et al., Biochemistry (1994) 33:6966). With the possibleexception of the coumarins, these fluorophores are also stable toreagents used for deprotection of peptides synthesized using FMOCchemistry (Strahilevitz et al., Biochemistry (1994) 33:10951). The t-BOCand α-FMOC derivatives of ε-dabcyl-L-lysine also can be used toincorporate the dabcyl chromophore at selected sites in a polypeptidesequence. The dabcyl chromophore has broad visible absorption and canused as a quenching group. The dabcyl group also can be incorporated atthe N-terminus by using dabcyl succinimidyl ester (Maggiora et al,supra). EDANS is a common fluorophore for pairing with the dabcylquencher in fluorescence resonance energy transfer experiments. Thisfluorophore is conveniently introduced during automated synthesis ofpeptides by using 5-((2-(t-BOC)-γ-glutamylaminoethyl) amino)naphthalene-1-sulfonic acid (Maggiora et al., J Med Chem (1992)35:3727). An α-(t-BOC)-ε-dansyl-L-lysine can be used for incorporationof the dansyl fluorophore into polypeptides during synthesis (Gauthier,et al., Arch Biochem Biophys (1993) 306:304). Like EDANS, itsfluorescence overlaps the absorption of dabcyl. Site-specificbiotinylation of peptides can be achieved using the t-BOC-protectedderivative of biocytin (Geahlen et al., Anal Biochem (1992) 202:68). Theracemic benzophenone phenylalanine analog can be incorporated intopeptides following its t-BOC or FMOC protection (Jiang, et al., Intl JPeptide Prot. Res (1995) 45:106). Resolution of the diastereomers isusually accomplished during HPLC purification of the products; theunprotected benzophenone can also be resolved by standard techniques inthe art. Keto-bearing amino acids for oxime coupling, aza/hydroxytryptophan, biotyl-lysine and D-amino acids are among other examples ofunnatural amino acids that can be utilized. It will be recognized thatother protected amino acids for automated peptide synthesis can beprepared by custom synthesis following standard techniques in the art.

[0045] A chemical tag also can be introduced by chemical modificationusing a reactive substance that forms a covalent linkage once havingbound to a reactive group of the target cross-over protein moleculeand/or one or more module containing peptide segments used to constructthe protein. For example, a target cross-over protein can includeseveral reactive groups, or groups modified for reactivity, such asthiol, aldehyde, amino groups, suitable for coupling the chemical tag bychemical modification (Lundblad et al., In: Chemical Reagents forProtein Modification, CRC Press, Boca Raton, Fla., (1984)).Site-directed mutagenesis of a cross-over protein module producedribosomally and/or via chemical synthesis also can be used to introduceand/or delete such groups from a desired position. Any number ofchemical tags including biotinylation probes of a biotin-avidin orstrepavidin system, antibodies, antibody fragments, carbohydrate bindingdomains, chromophores including fluorophores and other dyes, lectin,nucleic acid hybridization probes, drugs, toxins and the like, can becoupled in this manner. For instance, a low molecular weight hapten,such a fluorophore, digoxigenin, dinitrophenyl (DNP) or biotin, can bechemically attached to a target reactive group by employinghaptenylation and biotinylation reagents. The haptenylated polypeptidethen can be directly detected using fluorescence spectroscopy, massspectrometry and the like, or indirectly using a labeled reagent thatselectively binds to the hapten as a secondary detection reagent.Commonly used secondary detection reagents include antibodies, antibodyfragments, avidins and streptavidins labeled with a fluorescent dye orother detectable marker.

[0046] Depending on the reactive group, chemical modification can bereversible or irreversible. A common reactive group targeted in proteinsare thiol groups, which can be chemically modified by haloacetyl andmaleimide labeling reagents that lead to irreversible modifications andthus produce more stable products. For instance, reactions of sulfhydrylgroups with α-haloketones, amides, and acids in the physiological pHrange (pH 6.5-8.0) are well known and allow for the specificmodification of cysteines in peptides and polypeptides (Hermason et al.,In: Bioconjigate Techniques, Academic Press, San Diego, Calif., pp98-100, (1996)). Covalent linkage of a detectable label also can betriggered by a change in conditions, for example, in photoaffinitylabeling as a result of illumination by light of an appropriatewavelength. For photoaffinity labeling, the label, which is oftenfluorescent or radioactive, contains a group that becomes chemicallyreactive when illuminated (usually with ultraviolet light) and forms acovalent linkage with an appropriate group on the molecule to belabeled. An important class of photoreactive groups suitable for thispurpose is the aryl azides, which form short-lived but highly reactivenitrenes when illuminated. Flash photolysis of photoactivatable or“caged” amino acids also can be used for labeling peptides that arebiologically inactive until they are photolyzed with UV light. Differentcaging reagents can be used to modify the amino acids, such derivativesof o-nitrobenzylic compounds, and detected following standard techniquesin the art. (Kao et al., “Optical Microscopy: Emerging Methods andApplications,” B. Herman, J. J. Lemasters, eds., pp. 27-85 (1993)). Thenitrobenzyl group can be synthetically incorporated into thebiologically active molecule via an ether, thioether, ester (includingphosphate ester), amine or similar linkage to a hetero atom (usually O,S or N). Caged fluorophores can be used for photoactivation offluorescence (PAF) experiments, which are analogous to fluorescencerecovery after photobleaching (FRAP). Those caged on the ε-amino groupof lysine, the phenol of tyrosine, the γ-carboxylic acid of glutamicacid or the thiol of cysteine can be used for the specific incorporationof caged amino acids in the sequence. Alanine, glycine, leucine,isoleucine, methionine, phenylalanine, tryptophan and valine that arecaged on the α-amine also can be used to prepare peptides that are cagedon the N-terminus or caged intermediates that can be selectivelyphotolyzed to yield the active amino acid either in a polymer or insolution. (Patchornik et al., J Am Chem Soc (1970) 92:6333). Spinlabeling techniques of introducing a grouping with an unpaired electronto act as an electron spin resonance (ESR) reporter species may also beused, such as a nitroxide compound (—N—O) in which the nitrogen formspart of a sterically hindered ring (Oh et al., Science (1996)273:810-812).

[0047] Selection of a chemical tag for a given cross-over proteingenerally depends on its intended use. In particular, the chemicalligation methods and compositions of the invention can utilize achemical tag for application in a screening assay of the inventioncharacterized by binding of a cross-over protein to a target receptor.These include diagnostic assays, screening new compounds for drugdevelopment, and other structural and functional assays that employbinding of a cross-over protein to a target receptor. The methodsinclude the steps of contacting a receptor with one or more cross-overproteins obtained from a cross-over protein library, and identifying across-over protein from the library that is a ligand for the receptor inan assay characterized by detection of binding of the ligand to thereceptor. The methods preferably employ one or more of cross-overproteins having a detectable label, such as an unnatural amino acidincluding a chromophore. Of particular interest are chromophorescomprising an acceptor and/or donor moiety of an acceptor-donorresonance energy transfer pair. For cross-over proteins comprising atleast one chromophore, a preferred form of detection is fluorescencedetection. When a resonance energy transfer pair is represented, apreferred form of fluorescence detection is fluorescence resonanceenergy transfer detection (FRET). Screening methods of particularinterest involve contacting a target receptor with a cross-over proteinligand, where at least the cross-over ligand is labeled with one or morechromophores, followed by detection of ligand binding by fluorescencespectroscopy. The methods, compounds and compositions of the inventionare readily adaptable to high throughput screening.

[0048] When employed in a screening or diagnostic assay, a chemical tagcan be utilized as a handle to attach a cross-over protein of theinvention to a support matrix. Various reversible binding, covalentattachment, and/or cleavable linker moieties may be used for thispurpose to tether the molecule of interest to the support matrix. Apreferred support matrix is one amenable to storage, shipping, multiplexscreening and/or automated applications, such as chromatography columns,beads, multi-sample sheets such as nitrocellulose sheets, multi-wellplates and the like. In a preferred embodiment, the cross-over proteinsare attached to a solid support matrix in a spatially addressable array.For instance, a set of cross-over proteins representing a desiredcross-over ligation structure or group of structures may be logicallyarranged in spatially addressable multi-well microtiter plates (e.g., 96and/or 386 well microtiter plates) with a one or more cross-overproteins per well. These arrays may be assembled into larger array setsto increase information derivable from a screening and/or diagnosticassay.

[0049] Assays of particular interest employ receptors provided bytissues or cell preparations, synthetic preparations and the like.Receptors of particular interest are lipid membrane-bound receptorsgenerated by lipid matrix-assisted chemoselective chemical ligation asdescribed in co-pending application U.S. Serial No. [to be assigned]filed Aug. 31, 1998 (Attorney Docket No. GRFN-028/00US). Screening forbinding of a cross-over protein ligand comprising one or morechromophores to a target receptor is preferably performed in a FRETassay. Ligand binding can be measured by any number of methods known inthe art for FRET analyses, including steady state and time-resolvedfluorescence by monitoring the change in fluorescence intensity,emission energy and/or anisotropy, for example, through energy transferfrom a donor moiety to an acceptor moiety of the FRET system. (See,e.g., Wu et al., Analytical Biochem. (1994) 218:1-13). FRET assays allownot only distance measurements, but also resolution of the range ofdonor-to-acceptor distances. FRET also can be used to show that theligand and/or target receptor exists alternately in a singleconformational state, or with a range of donor-to-acceptor distanceswhen in a different state, such as when bound to a ligand. More than onedonor-acceptor pairing may also be included.

[0050] For FRET assays, the cross-over protein ligand is designed tocontain at least one chromophore of a donor-acceptor system. The donormolecule is always a fluorescent (or luminescent) one for detection. Theacceptor molecule can be either fluorescent or non-fluorescent. Thus fora donor-acceptor system, at least two chromophores are provided: thefirst is provided by the cross-over ligand; the second can be providedby the receptor, a matrix to which the receptor and/or ligand isattached and/or embedded such as a lipid membrane, or by one or more ofa second ligand for the receptor and/or cross-over ligand.

[0051] When choosing a chromophore donor-acceptor pair for FRET,positioning of the first chromophore in a target cross-over proteinligand is selected to be within a sufficient distance of a secondchromophore to create a donor-acceptor fluorescence resonance energytransfer system. For instance, energy transferred from the donor to anacceptor involves coupling of dipoles in which the energy is transferredover a characteristic distance called the Forster radius (R_(o)), whichis defined as the distance at which energy transfer efficiency is 50%(i.e., distance at which 50% of excited donors are deactivated by FRET).These distances range from about 10 to 100 Angstroms (Å), which iscomparable to the diameter of many proteins and comparable to thethickness of membranes. Intrinsic tryptophan or tyrosine sometimes maybe used as chromophores in distance measurements, but in most cases theForster distance is limited to above 30 Å. However, an acceptor moleculecomprising clusters of acceptors with high molar absorption coefficientfor each acceptor may achieve a further extension of Forster distance.Thus average distances over 100 Å can be measured. As the Forsterdistances can be reliably calculated from the absorption spectrum of theacceptor and the emission spectrum of the donor, FRET allowsdetermination of molecular distances. Once the Forster distance isknown, the extent of energy transfer can be used to calculate thedonor-to-acceptor distance.

[0052] Donor-acceptor chromophores applicable for biological molecules,and for which Forster distances are known when paired, include but arenot limited to the following chromophores: ANAI (2-anthracenceN-acetylimidazole); BPE (B-phycoerythrin); CF (caboxyfluoresceinsuccinimidyl ester); CPM(7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin); CY5(carboxymethylindocyanine-N-hydroxysuccinimidyl ester, diI-C₁₃,1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine; diO-C₁₄,3,3′-ditetradecyloxacarbocyanine); DABM(4-dimethylaminophenylazo-phenyl-4′-maleimide); DACM((7-(dimethylamino)coumarin-4-yl)-acetyl); DANZ (dansylaziridine); DDPM(N-(4-dimethylamino-3,5-dinitrophenyl)maleimide); DMAMS(dimethylamino-4-maleimidostilbene); DMSM(N-(2,5-dimethoxystiben-4-yl)-maleimide); DNP (2,4-dinitrophneyl); -A(1,N⁶-ethenoadenosine); EIA (5-(iodoacetetamido)eosin); EITC (eosinthiosemicarbazide); F₂DNB (1,5-difluro-2,4′-dinitrobenzene); F₂DPS(4,4′-difluoro-3,3 ′-dinitrophenylsulfone); FITC(fluorescein-5-isothiocyanate); FM (fluorescein-5-maleimide); FMA(fluorescein mercuric acetate); FNAI (fluorescein N-acetylimidazole);FTS (fluorescein thiosemicarbazide); IAANS(2-(4′-iodoacetamido)aniino)naphthalene-6-sulfonic acid); IAEDANS(5-(2-((iodoacetyl)amino)ethyl)amino)-naphthlene-l-sulfoni acid); IAF(5-iodoacetamidofluorescein); IANBD(N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole);IPM (3(4-isothiocyanatophenyl)7-diethyl-4-amino-4-methylcoumarin); ISA(4-(iodoacetamido)salicylic acid); LRH (lissaminerhodamine); LY (Luciferyellow); mBBR (monobromobimane); MNA ((2-methoxy-1-naphthyl)-methyl);NAA (2-naphthoxyacetic acid); NBD (7-nitro-2,1,3-benzoxadiazol-4-yl);NCP (N-cyclohexyl-N′-(1-pyrenyl)carbodiimide); ODR (octadecylrhodamine);PM (N-(1-pyrene)-maleimide); SRH (sulforhodamine); TMR(tetramethylrhodamine); TNP (trinitrophenyl); TR (Texas red); BODIPY((N1-B)-N1′-(difluoroboryl)-3,5′-dimethyl-2-2′-pyrromethene-5-propionicacid, N-succinimidyl ester); and lanthanide-ion-chelates such as aniodoacetamide derivative of the Eu3+-chelate of N-(p-benzoicacid)diethylenetriamine-N,N′,N′-tetraacetic acid (DTTA).

[0053] Since energy transfer measurement is most sensitive to distancevariation when donor-acceptor separation is close to their Forsterdistance, the molecule comprising the first chromophore of adonor-acceptor pair system is selected or engineered so that the firstand second chromophores approach or are at the Forster distance. Table 1shows some typical Forster distances of donor-acceptor pairs. TABLE 1Donor Acceptor Forster Distance (Å) Fluorescein Tetramethyllrhodamine 55IAEDANS Fluorescein 46 EDANS DABCYL 33 Fluorescein Fluorescein 44 BODIPYFL BODIPY FL 57

[0054] Extensive compilations of Forster distances for variousdonor-acceptor pairs and their specific applications in FRET analysis ofbiological molecules including peptides, proteins, carbohydrates andlipids are well known in the art. (See, e.g., Wu et al., supra; Berlmanet al., (1973) Energy Transfer Parameters of Aromatic Compounds,Academic Press, New York; Van der Meer et al., (1994) “Resonance EnergyTransfer Theory and Data,” VCH Publishers; dos Remedios et al., J MuscleRes Cell Motility (1987) 8:97; Fairclough et al., Meth Enzymol (1978)48:347). These Forster distances are used as a general guide whenselecting a particular donor-acceptor pair.

[0055] In addition to selecting donor and acceptor moieties that are inclose proximity (typically 10-100 Å) and approach or are at the Forsterdistance, the FRET chromophore pairs are selected so that the absorptionspectrum of the acceptor overlaps the fluorescence emission spectrum ofthe donor, and the donor and acceptor transition dipole orientations areapproximately parallel. Moreover, for anisotropy assays the chromophoresare preferably positioned so that tumbling of the donor or acceptormoiety is minimized. An advantage of reducing chromophore tumbling isincreased sensitivity in FRET detection by reducing background noise inthe spectrum.

[0056] For most applications, the donor and acceptor dyes are different,in which case FRET can be detected by the appearance of sensitizedfluorescence of the acceptor (acceptor enhancement), by quenching ofdonor fluorescence (donor quenching), or fluorescence polarization(anisotropy). When the donor and acceptor are the same, FRET istypically detected by anisotropy. For instance, donor quenching(quenching of fluorescence) can be used to detect energy transfer.Excitation is set at the wavelength of donor absorption and the emissionof donor is monitored. The emission wavelength of donor is selected suchthat no contribution from acceptor fluorescence is observed. Thepresence of acceptor quenches donor fluorescence. A wide variety ofsmall molecules or ions act as quenchers of fluorescence, that is, theydecrease the intensity of the emission. These substances include iodide,oxygen, chlorinated hydrocarbons, amines, and disulfide groups. Theaccessibility of fluorophores to quenchers is widely used to determinethe location of probes on macromolecules, or the porosity of cross-overproteins or target receptor to the quenchers.

[0057] Acceptor enhancement detection techniques can be used when anacceptor is fluorescent, and its fluorescence intensity is enhanced whenenergy transfer occurs (with excitation into the donor). This providesadditional methods to visualize energy from a fluorescence spectrum. Inan emission spectrum, one excites at the wavelength of donor absorptionand observes the intensity increase of acceptor. In an excitationspectrum, one sets detection at the acceptor emission wavelength andobserves enhancements of intensity at a wavelength range where donorabsorbs.

[0058] Anisotropy (or fluorescence polarization) analysis using FRET isof particular interest. The polarization properties of light and thedependence of light absorption on the alignment of the fluorophores withthe electric vector of the incident light provide the physical basis foranisotropic measurements. Fluorescence probes usually remain in theexcited state from 1 to 100 nanoseconds (ns), a duration called thefluorescence lifetime. Because rotational diffusion of proteins alsooccurs in 1-100 ns, fluorescence lifetimes are a favorable time scalefor studies of the associative and/or rotational behavior ofmacromolecules. Other probes may be employed that remain in the excitestate longer than 1-100 ns, such as those that remain in excited statefor several 100 μs. When a sample of a cross-over protein systemcomprising an appropriate donor-acceptor chromophore pair is illuminatedwith vertically polarized light, the emission can be polarized. Whenenergy transfer occurs between the same molecules in identicalenvironments, fluorescence intensity or lifetime does not change. Theanisotropy on the other hand may change due to likely change inchromophore orientation. For example, binding of cross-over proteinligand may alter the rotational motions of a receptor for the ligandduring the lifetime of the excited state, where slower rotationaldiffusion results in higher polarization of the emitted light. Hence, ifa receptor binds a ligand that induces a conformational change in thechromophore orientation by decreasing its rotational rate, theanisotropy increases. Thus, by means of fluorescence, and in particular,measurements of fluorescence polarization (or anisotropy), it ispossible to measure rotational motions of a cross-over protein ligandand/or receptor for the ligand.

[0059] Homogenity and structural identity of the desired covalentligation product can be confirmed by any number of means including highperformance liquid chromatography (HPLC) using either reverse phase orion exchange columns, mass spectrometry, crystallography and nuclearmagnetic resonance (NMR). Characterization of synthetic peptides alsocan be performed by a combination of amino acid analysis and massspectrometry. The positions of the modifications and deletions, ifpresent, can be identified by sequencing with either chemical methods(Edman chemistry) or tandem mass spectrometry.

[0060] The chemical ligation approaches described herein is extendableto the combination (cross-over) of as many segments or functionalmodules as is possible based upon chemical ligation sites present in thesequence. For example, native chemical ligation at naturally occurringcysteine residues can be adapted to other regions devoid of cysteines byintroducing cyteines at other positions. The same is true for otherligation chemistries,. i.e., chemoselective reactive groups can beengineered into a desired position so as to facilitate site-directedligation. The chemical ligation approach is applicable to many proteinsystems. Combination of segments from regions of related proteins withanalogous segments of related proteins is advantageous because itcapitalizes on the diversity of a class of proteins, creating newproteins with new properties. These new properties may be novel (unknownin either parent protein) or more restricted (a subset of the bindingproperties of the parent proteins). Either of these new types ofproperties are desirable.

[0061] Of particular interest are classes of proteins that havetherapeutic potential, and have functional modules that are readilyaccessible by chemical synthesis. A number of classes of proteins areknown and include the chemokines; macrophage migration inhibitoryfactor; other cytokines; trefoil peptides; growth factors; proteaseinhibitors; and toxins. For example, these proteins are ligands forparticular receptors.

[0062] Protein ligands of particular interest are those which arecapable of binding to various receptors such as enzyme-linked receptors,fibronectin-like receptors, the seven transmembrane receptors, and theion channel receptors, including the tryosine and serine-theroninekinases, and gluanylate cyclase families of enzyme-linked receptors.Examples of the tyrosine kinase family of receptors include epidermalgrowth factor, insulin, platelet-derived growth factor, and nerve growthfactor. Examples of the serine kinase family of receptors include growthfactor β-family. Examples of the guanylate cyclase family includes thosereceptors that generate cyclic GMP (cGMP) in response to atrialnatriuretic factors. Examples of the seven-transmembrane receptorsinclude those membrane proteins that bind catecholamines, histamines,prostoglandins, etc., and the opsins, vasopressin, chemokine andmelanocortin receptors. Examples of the ion channel receptors arerepresented by the ligand- and voltage-gated channel membrane proteinreceptors, and include the acetylcholine activated sodium channels,glycine and gamma-aminoisobutyric acid activated chloride channels, andserotonin and glutamate activated calcium channels, and the family ofcyclic nucleotide-gated channels (cAMP and cGMP), and the family ofinositol 1,4,5-triphosphate (IP3) and the cyclic ADP-ribose receptorsthat modulate calcium storage. One of ordinary skill in the art willrecognize that nucleic acid and/or amino acid sequences for the aboveand additional receptors and their protein ligands can be identified invarious genomic and protein related databases. Examples of publiclyaccessible databases include as GenBank (Benson et al., Nucleic AcidsRes. (1998) 28(l):1-7, USA National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md., USA), TIGR Database (The Institute for GenomicResearch, Rockville, Md., USA) Protein Data Bank (Brookhaven NationalLaboratory, USA), and the ExPASy and Swiss-Protein database (SwissInstitute of Bioninformatics, Geneve, Switzerland).

[0063] Of particular interest are protein classes or families ofproteins amenable to native chemical ligation, and thus having naturallyoccurring conserved cysteine residues, or residues locations into whichcysteine residues can be introduced. Examples include chemokines,agouti-related proteins, and the sex determining proteins DSX and DMT1.

[0064] Preferred cross-over proteins of the invention include ligandsfor the chemokine receptors and melanocortin receptors. Chemokinescomprise a large family of structurally homologous cytokines,approximately 8 to 10 kD in size. These molecules share the ability tostimulate leukocyte movement (chemokinesis) and directed movement(chemotaxis). All of these molecules contain two internal disulfideloops. Chemokines have been classified into subfamilies, based onwhether the two amino terminal cysteine residues are immediatelyadjacent (cys-cys or CC) or separated by one amino acid (cys-X-cys orCXC) or three amino acids (cys-XXX-cys or CXXXC) based on spacingproximal for the amino terminus. The chemokines fall into two majorsubclasses: (1) CC chemokines, which generally act on leukocytesincluding monocytes, T-cells, eosinophils, and basophils; and (2) CXCchemokines, which are primarily involved in acute inflammation andneutrophil activation. Members of the CXC, α-chemokine or 4 q family mapto human chromosome 4 q12-21. The chemokine protein family comprisesmore than 65 proteins identified to date. Some of these include, membersof the CXC chemokine group, such as Platelet Factor 4 (PF4), PlateletBasic Protein (PBP), Interleukin-8 (IL-8), Melanoma Growth StimulatoryActivity Protein (MGSA), Macrophage Inflammatory Protein 2 (MIP-2),Mouse Mig (m119), Chicken 9E3 (or pCEF-4), Pig Alveolar MacrophageChemotactic Factors I and II (AMCF-I and -II), Pre-B Cell GrowthStimulating Factor (PBSF) (Stromal Cell-Derived Factor 1) (SDF-1), andIP10, a gamma-interferon induced protein. Members of the CC chemokinegroup, or b-chemokine or 17 q family map to human chromosome 17q11-32(murine chromosome 11)., and include Monocyte Chemotactic Protein 1, 2and 3 (MCP−1, −2.−3), Macrophage Inflammatory Protein 1 alpha, beta andgamma (MIP-1-alpha, MIP-1-beta, and MIP-1-gamma), MacrophageInflammatory Proteins 3, 4 and 5 (MIP-3, MIP-4, and MIP-5), LD-78 beta,RANTES, Eotaxin, 1-309 (also known, in mouse, as TCA3), mouse proteinC10, and mouse protein Marc/FIC. In addition to the CC and CXC familiesof chemokines, other groups have been identified including the “C”chemokines that are encoded by the genes SCYC1 ans SCYC2, the “CXXXC”chemokines encoded by SCYD1, and virus-encoded chemokines from virusessuch as Marek's disease virus (Gallid herpesvirus 1) (Eco Q protein),stealth virus (unclassified), Kaposi's sarcoma-associated herpes-likevirus (vMIP-IA) and (vMIP-I), Kaposi's sarcoma-associated herpes-likevirus (vMIP-1B) and (vMIP-II), malluscum contagiosum virus (MC148R),murine cytomegalovirus (MCK-1 (ORF HJ1), human herpesvirus-6 variant Astrain (EDRF3), and human herpesvirus-6 variant B strain (Z29) (CB11R).

[0065] Many of the chemokines are strongly expressed during the courseof a number of pathophysiological processes including autoimmunediseases, cancer, atherosclerosis, and chronic inflammatory diseases.The biological activities of chemokines are mediated by specificreceptors and also by receptors that bind several other proteins. Forinstance, the chemokine receptors include the CCR1, CCR2, CCR3, CCR4,CCR5, CCR6, CCR8, CXCR1, CXCR2, CXCR3, and CXCR4 chemokine receptors.Also included are the P-chemokine receptors and the unclassifiedchemokine receptors. There also are several receptors with homology tothe chemokine receptors. For example, ligands for CCR1 include RANTES,MIP-1α, MCP-2, MCP3. Ligands for CCR2 include MCP-1, MCP-2, MCP-3, andMCP-4. Ligands for CCR3 include Eotaxin, eotaxin-2, RANTES, MCP-2,MCP-3, and MCP-4. Ligands for CCR4 include TARC, RANTES, MIP-1α, andMCP-1. Ligands for CCR5 include RANTES, MIP-1α, and MIP-1β. Ligands forCCR6 include LARC/MIP-3α/exodus. Ligands for CCR7 include ELC/MIP-3β.Ligands for CCR8 include I-309. Ligands for CXCR1 include IL-8 andGCP-2. Ligands for CXCR2 include IL-8, GRO-α/β/γ, NAP-2, ENA78 andGCP-2. Ligands for CXCR3 include IP10 and Mig. Ligands for CXCR4 includeSDF-1. Ligands for CXCR5 include BCA-1/BLC. For example, SDF-1α, a CXCchemokine, is the natural ligand for CXCR4 (also called fusin, LESTR andHUMSTR). T-tropic HIV strains bind to CD4 and then depend on subsequentbinding to the CXCR4 receptor for entry into cells. SDF-1 thus has thepotential to block HIV binding to CXCR4. The chemokine family ofproteins are thus prime targets for development of lead compounds incharacterizing and treating such disorders.

[0066] The characteristic pattern of cysteine residues in chemokines isparticularly well suited to the systematic production of focused sets ofmodular hybrid chemokine analogues by native chemical synthesis.Chemokines represent a class of proteins with varied overlappingreactivity and functions, both at the receptor and cell levels. Severalchemokine structures have been solved by NMR and X-ray crystallography.The three-dimensional structures are highly homologous and represent aninvariant peptide backbone or scaffolding. The structures also show ahighly conserved set of amino acids forming the hydrophobic core.Because of the structural homology across approximately 65 chemokines(to date), the various segments of the chemokines are particularly wellsuited for swapping of functional modules (i.e., cross-over synthesis)between each other to construct novel chemokine libraries, and identifydifferent activities related to structure and function.

[0067] Except for the CXC chemokine PBSF, consensus patterns of the CXCchemokines have been shown, as illustrated below beginning from thecysteines of the N-terminus:

[0068]_(n)X(1,8)-C-X-C-[LIVM]-X(5,6)-[LIVMFY]-X(2)-[RKSEQ]-X-[LIVM]-X(2)-[LIVM]-X(5)-[SAG]-X(2)-CX(3)-[EQ]-[LIVM]-X(2)-X(9,10)-CL-[DN]

[0069] Consensus patterns of the CC chemokines also have been shown, asillustrated below beginning from the cysteines of the N-terminus:

[0070]_(n)X(1,9)-C-C-[LIVMFYT]-X(5,6)-[LIVM]-X(4)-[LIVMF]-X(2)-Y-X(2,3)-[GSTN](2)-X(1,2)-C-X(3,4)-[SAG]-[LIVM]-X(2)-[FL]-X(5)- [RKTMF]-X(2)-C

[0071] Since chemokines contain cysteine sites which are amenable tonative chemical ligation, the modular chemokines can be readilysynthesized in two or four segments without the need to introduceadditional cysteines or use other ligation methods. As an example,cross-over chemokines produced using a two segment approach have anN-terminal segment from one chemokine and a C-terminal segment fromanother as shown in scheme (1) below. The novel proteins are assessedfor different properties contributed from the original, parentchemokines.

[0072] Where the native chemokine sequences 1 and 2, where A, B, R, andS are arbitrary amino acids determined in the naturally occurringchemokine, and each can by synthesized by native chemical ligation oftwo segments, and C represents a Cysteine, the site which is amenable tonative chemical ligation.

[0073] In accordance with Scheme 1, the N-terminal segment of chemokine1 (fictitiously consisting of all A amino acids) can be ligated to theC-terminal segment of chemokine 1 (fictitiously consisting of cysteine(C) followed by all B amino acids). Likewise, chemokine 2 can besynthesized by the ligation of the N-terminal segment of chemokine 2 tothe C-terminal segment of chemokine 2. Each chemokine folds into thenatural, biologically-active protein. The cross-over chemokine (1/2) ismade by ligating the N-terminal segment of chemokine I to the C-terminalsegment of chemokine 2. Likewise, an additional unique crossoverchemokine, chemokine (2/1) is made by ligating the N-terminus ofchemokine 2 to the C-terminal segment in chemokine 1. The nativechemical ligation can be applied between any residue and a cysteine.Typically, chemokines contain four cysteines and therefore can be madein five segments (four native ligations). As noted above, cysteines alsomay be designed into the structure to permit alternative ligation sitesamenable to native ligation chemistry. Additionally, other types ofligation permit assembly of chemokines and other proteins at othersites.

[0074] The melanocortin family of receptor-ligands also are examples ofproteins amenable to cross-over synthesis as exemplified above for thechemokines. For instance, the melanocortin receptors include themelanocyte melanocortin receptor (MC1R), MC2R (adrenocortical ACTHreceptor), MCR3, MCR4 and MCR5 receptors. Ligands for variousmelanocortin receptors include agouti protein (AGP) and agouti-relatedproteins (AGRP). Of particular interest are analogues of AGRP, includingminimized agouti-related proteins (MARP) as disclosed in Thompson etal., co-pending provisional patent application U.S. Ser. No. 06/079,957.

[0075] The cross-over proteins and libraries can be used in a variety oftherapeutic applications. Preferred hybrid proteins are those comprisingcross-over members of the chemokine family, and analogs derivedtherefrom. The modular chemokines of the invention may be used in avariety of therapeutic areas, including inflammation and infectiousdiseases such as AIDS, as well as in indications for hematopoiesis andchemoprotection. Modified derivatives of the native compounds also havebeen shown to effectively block the inflammatory effects of RANTES.Accordingly, they are useful for the treatment of asthma, allergicrhinitis, atopic dermatitis, atheroma/atheroschleosis, and rheumatoidarthritis. Chemokines also have been shown to inhibit HIV-1 infection invitro. Additional cross-over proteins and libraries of interest arecross-over members of agouti protein ligands for the melanocortinreceptor family, including AGP and MARP that are useful for modulatingsatiety in a mammal or a disease state such as a wasting syndrome in amammal including HIV wasting syndrome, cachexia, or quorexia. Forinstance, cross-over agouti proteins find use as leads in treatingfeeding disorders, obesity, and other disorders related to hypothalamiccontrol of feeding. A wasting syndrome is an illness characterized bysignificant weight loss accompanied by other indicia of poor health,including poor appetite, gut disorder, or increased metabolic rate.Wasting syndromes include, but are not limited to, the wasting syndromeafflicting some patients diagnosed with Acquired Immune DeficiencySyndrome (AIDS) and various cancers. As methods of treating othersymptoms of diseases such as AIDS progress, the incidence of wastingsyndrome as the cause of death increases. Improved prophylaxis andtreatment for HIV wasting syndrome is required (Kravick et al., Arch.Intern. Med. (1997) 157:2069-2073). Anorexia and cachexia are well-knownresults of cancer that contribute to morbidity and mortality (Simons etal, Cancer (1998) 82:553-560; Andrassy et al., Nutrition (1998)14:124-129). The reasons for the significant weight loss are multipleand may be directly related to the tumor, such as increased metabolicrate, but also include decreased intake due to poor appetite or gutinvolvement. Further, excessive leptin-like signaling may contribute tothe pathogenesis of wasting illness (Schwartz et al., Pro. Nutr. Soc.(1997) 56:785-791).

[0076] The invention further includes a pharmaceutical compositioncomprising a cross-over protein of the invention, such as one derivedfrom a cross-over protein library of the invention. Also provided arekits having a cross-over protein of the invention, and/or or produced bya method(s) of the invention.

[0077] In applying the compounds of this invention to treatment of theabove conditions, administration of the active compounds and saltsdescribed herein are preferably administered parenterally. Parenteraladministration is generally characterized by injection, eithersubcutaneously, intramuscularly or intravenously, and can includeintradermal or intraperitoneal injections as well as intrasternalinjection or infusion techniques. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol or the like. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, such as for example, sodiumacetate, sorbitan monolaurate, triethanolamine oleate, etc.

[0078] For parenteral administration there are especially suitableaqueous solutions of an active ingredient in water-soluble form, forexample in the form of a water-soluble salt, or aqueous injectionsuspensions that contain viscosity-increasing substances, for examplesodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired,stabilizers. The active ingredient, optionally together with excipients,can also be in the form of a lyophilisate and can be made into asolution prior to parenteral administration by the addition of suitablesolvents. Solutions such as are used, for example, for parenteraladministration can also be used as infusion solutions. A more recentlydevised approach for parenteral administration employs the implantationof a slow-release or sustained-release system, such that a constantlevel of dosage is maintained. See, e.g., Higuchi et al., U.S. Pat. No.3,710,795, which is hereby incorporated by reference.

[0079] The percentage of active compound contained in such parentalcompositions is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject. However,percentages of active ingredient of 0.01% to 10% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. Preferably thecomposition will comprise 0.02-8% of the active agent in solution.

[0080] There are more than 65 known chemokines, and additional newsequences are being added to public genome databases at a rapid rate.Ligands for other therapeutically important receptors also are beingidentified and characterized at a significant rate. Construction ofcross-over protein libraries can be used for the rapid conversion ofgenomic data into high-purity novel proteins that can be usedcontiguously, and also can be used for the preparation of a wide rangeof analogues by chemical modification, such as N-terminal modification.Modular protein libraries can be used to define proteinstructure-activity relationships and to identify new lead compounds fortreatment of mammalian disorders. The construction of modular cross-overprotein libraries also can be used to improve the therapeutic utility ofa native protein by, for example, improving its binding affinity andspecificity, or by increasing its circulating half life. The modularhybrid approach described here has widespread applications in analyzingimportant structural determinants in other classes of molecules. Thenovel molecules are useful for in vitro studies of viral infection andfor therapies based on administration and over-expression of mutants oranalogs of these chemokines. Modular synthesis of cross-over chemokineshaving a combination of cross-over activities obtained from CC or CXCchemokines can be used as novel therapeutic leads and to assess thestructural basis of properties such as folding, stability, catalyticactivity, binding. and biological action. The dual agonist activities ofthe modular chemokines are particularly suited as antagonist and/oragonist against HIV infection. Cross-over melanocortin receptor-specificligands such as AGP protein, AGRP and MARP also are examples oftherapeutic proteins accessible by the methods of the invention that canbe used as novel therapeutic leads. Libraries of chemokines and agouticross-over proteins generated by the chemical synthesis methods of theinvention represent compound libraries having unprecedented focuseddiversity, high yield and purity, where the product is free of cellularcontaminants.

[0081] Purity and yield are important for screening and therapeuticpurposes. Very often quantity of a compound in a library is a limitingfactor for the type and number of screening assays that can be employed.For example, the detection method typically is limited in part by theamount of a compound obtained from a library. In addition, purity is ofcritical important for efficacious screening of compounds in biologicalassays, to avoid skewed results contributed by impurities. Of coursepurity and yield is necessary when a cross-over protein is utilized fortherapeutic purposes, so as to minimize contaminants and provideunlimited access to high quality and certified product. Since thelibraries of the invention can be generated by chemical synthesis andligation, yield and purity can be controlled.

[0082] The methods and compositions of the invention also can beexploited in screening and diagnostic assays, and are particularlyamenable to resonance energy transfer assays employing FRET analyses.This includes access to donor-acceptor chromophore systems that can beused as a qualitative or a quantitative tool to detect and characterizeinteractions between a receptor-ligand system of interest. Theprinciples and applications of employing resonance energy transfersystems are many and well known (Wu et al., supra). For instance, thecross-over protein ligands can be simultaneously constructed and labeledvia native chemical ligation to create a chromophore donor/acceptorsystem that enables detection through FRET. Since measurement of energytransfer is based on fluorescence detection, the assays are highlysensitive and can be used to detect ligand binding. Since the time scaleof resonance energy transfer is on the order of nanoseconds, manyprocesses including slow conversion of conformers that are time-averagedin other techniques can be resolved. This approach can be used to inferthe spatial relation between donor and acceptor chromophores to obtainstructural information, including ligand-induced conformational changes.In addition to data acquisition with a conventionalspectrophotofluorometer, the FRET methods can be adapted for multiple invitro and in vivo assays including liquid chromatography,electrophoresis, microscopy, and flow cytometry etc. Thus, the presentinvention can be used for both in vitro and in vivo assays. The methodalso can be applied as a simple diagnostic tool, as well as used in thestudy of membrane structure and dynamics, or extend it to molecularinteractions on cell surfaces or in single cells.

[0083] The following examples are presented to illustrate the inventionand are not intended to be limiting.

EXAMPLES Example 1

[0084] Identification of Functional Protein Modules for Synthesis ofCross-over Chemokine Libraries

[0085] Chemokine patterns are compared on a linear amino acid sequencelevel and on a three-dimensional structural level to identify functionalprotein modules for the modular synthesis of cross-over chemokinelibraries. Functional protein modules corresponding to homologousregions among the native chemokines are identified by alignment ofsegments of RANTES, SDF-1α, and the virally encoded chemokines vMIP-Iand vMIP-II (See FIG. 3). Macrophage Derived Chemokine (MDC) and theKaposi's sarcoma-associated herpes virus (KSHV) vMIP-I and II chemokinesalso are compared. Sequence alignment of RANTES, SDF-1α and the viralchemokines against the RANTES three-dimensional structure (BrookhavenProtein Databank, Brookhaven National Labs, NY) using LOOK® software(Molecular Applications Group, Palo Alto, Calif.) identified sections ofsequences that correlated with functional sections relative to thefolded chemokines. On a sequence level the chemokines are found to bedivided into segments by the cysteines, typically at positions 8, 9, 34and 50 relative to the functional molecules (positions 10, 11, 34 and50, respectively, as depicted in FIG. 3). Each of the interveningsegments is found to provide some part of overlapping binding sites forvarious receptors. The N-terminal segment (residues 1-8) has been shownto be important for receptor activation; truncation of the N-terminalsegment can yield antagonists that bind but do not signal (e.g., RANTES,Arenzana-Selsdedos et al., Nature (1996) 383: 400). The second segment(residues 8-9) identified contains either 0, 1, or 3 amino acids.Although this segment is short, the CC-chemokines (zero amino acids inthis segment) and the CXC-chemokines (one amino acid in this segment)bind to two different sets of receptors with no overlap between them.The third segment (residues 9-34) identified can be divided into twodistinct regions. Segment (residues 9-22) interacts with the7-transmembrane G-protein receptors. The segment (residues 23-34) isidentified as comprising the dimer interface based upon comparison ofthe three-dimensional structures of CXC chemokines like IL8. The fourthsegment (residues 35-50) is identified as comprising a central betastrand which contributes to the hydrophobic core and a region (43-49)which also interacts with the 7-transmembrane G-protein receptors. ForIL-8 and GRO gamma, the regions 9-22 and 43-49 also have been shown tobe important for determining binding to different receptors (Hammond etal., supra). The fifth segment (residues 51-75) is identified ascontaining a C-terminal helix which contributes to the hydrophobic coreand contains a heparin-binding domain. Crossing-over the binding regionsbased upon location of the cysteines, permits the separation of the fourregions most important for binding to the 7-transmembrane G-proteinreceptors: residues (1-8), (8-9), (9-23), and (43-49).

[0086] In addition, an asparagine to alanine substitution at position 33of a synthetic SDF-1α has been shown to be a more potent activator ofchemotaxis compared to the native SDF-1α sequence. This indicates thatthe N33A substitution improves receptor-mediated activation. Thesubstituted amino acid precedes the central cysteine that approximatelyseparates the chemokine into halves. Alignment of the CC chemokines inLOOK® with the seven-color scheme reveals that the N-terminus and thetwo amino acids before the central cysteine appeared to be relativelyunique. The substitution at position 33 also may effect a putativeswitch for activating the receptor and/or agonist binding. Constructionof modular chemokines comprising functional modules from RANTES andSDF-1α are used to characterize receptor activation andagonist/antagonist design.

Example 2

[0087] Modular Synthesis Of Cross-Over Chemokine Libraries

[0088] The cross-over chemokine libraries are chemically synthesizedusing solid phase and native chemical ligation at Xxx-Cys residues.SDF-1α has been synthesized by stepwise solid phase peptide chemistry(Bleul et al., Nature (1996) 382:829) and (Oberlin et al., Nature (1996)382:833). SDF1-α also has been synthesized by native chemical ligation.These techniques are employed to construct MPBV/MPAV, RANTES/SDF-1αcross-over chemokines discussed in the examples that follow. See in-situneutralization Boc-peptide synthesis as described in Schnolzer et al.,Int. J Peptide Protein Res. (1992) 40:180; chemical synthesis and nativeligation of proteins as described in Dawson et al., supra; Muir, (1993)Current Opinion Biochem. 4:420; Canne et al., J. Am. Chem. Soc (1995)117:2998; Lu et al., J. Am. Chem. Soc. (1996) 118:8518; and Lu et al.,Biochemistry (1997) 36(4):673; and thioester resins for Boc-peptidesynthesis as described in Hono et al., Chem. Soc. Jpn. 64, 111 (1991);Tam et al., Proc. Natl. Acad. Sci USA (1995) 92:12485; and Canne et al.,Tetrahedron Lett. (1995) 36:1217; and chemokines and assays as describedin Baggiolini et al., Cytokine (1991) 3:165; Oppenheim, Adv. Exp. Med.Biol. (1993) 351:183; Sykes et al., Science (1994) 264:90; Clark-Lewiset al., J. Biol. Chem. (1994) 269:16075; and Hromas et al., Blood (1997)89(9):3315.

[0089] Briefly, chemical synthesis is preformed using Boc protectedamino acids obtained from AnaSpec (San Jose, Calif.), Bachem California(Torrance, Calif.), Bachem (Philadelphia, Pa.), NovaBiochem (San Diego,Calif.), Peninsula Laboratories (Belmont, Calif.) or PeptidesInternational (Louisville, Ky.). Protected amino acids as follows:Arg(Tos), L-Asp(OChx), Asn(Xan), L-Glu(OChx), His(DNP), Lys(2ClZ),Ser(Bz), Thr(Bz), Tyr(2BrZ). DMF and DCM are HPLC grade and used asreceived. Trifluoroacetic acid is obtained from HaloCarbon (River Edge,N.J.).

[0090] Peptides are synthesized on a modified ABI430A instrument usingin situ neutralization boc chemistry protocols. C-terminal segments areprepared on-OCH2Pam resins (ABI, Foster City, Calif.). N-terminalsegments are prepared on α-thio-carboxylate-resin. Standard HF cleavageprotocols are employed following N-terminal Boc removal and drying ofthe resin. HPLC purification is performed on Rainin HPLCs (Woburn,Mass.) using Vydac C4 (4.6 and 25 mm) or Dynamax C4 (4.6 mm or 2 in)columns with gradient elution (A: 0.1% TFA, B: ACN, 0.1% TFA).Electrospray mass spectrometry is performed on a Sciex API1 (PE-Sciex).

[0091] Ligation is performed at 4 mM peptide concentration in 6Mguanidine. 0.1M phosphate, pH=7.0 in the presence of 33 mM thiophenol(Fluka, Switzerland) at room temperature. Ligation is monitored by HPLCand typically complete within 24 hours. Ligation is followed by HPLCpurification and lyophilization as described above.

[0092] Folding of synthetic chemokines is conducted as follows. Afterpurification, the full-length peptide is reduced at 1.0 mg/mL in 8M urea(Fluka, Switzerland), 0.1 M TRIS (Fluka, Switzerland), 5.37 mM EDTA(Fluka, Switzerland), pH=8.6 in the presence of 100 mM 2-mercaptoethanol(Fluka, Switzerland). Reduction occurs under a nitrogen atmosphere at40° C. for one hour. After complete reduction, the mixture is dilutedinto the same buffer at 0.2 mg/mL with 18.7 mM oxidized glutathione(Sigma Chemical, St. Louis, Mo.). The solution is dispensed into aSpectrum Spectra/Por *7 dialysis membrane (Houston, Tex.) (MWCO=3500)and the bag placed in 1.0 L of initial dialysis buffer of 8M urea, 0.1MTRIS, 1 mM EDTA, 3 mM 2-mercaptoethanol, 1.3 mM oxidized glutathione,pH=8.6. Then, over a period of two days, 4 liters of 2M urea, 0.1M TRIS,pH=8.6 is pumped into the vessel containing the dialysis bag. Folding ismonitored by HPLC and mass spectrometry and is usually complete after 3buffer changes (3 liters).

[0093] Alternatively, full length peptide is reduced directly from theligation conditions at 1 mg/mL in 6M guanidine.HCl (Fluka, Switzerland),0.1M TRIS, pH=8.5 in the presence of 100 mM 2-mercaptoethanol. Afterpurification on reversed phase HPLC and lyophilization, the peptide isoxidized at 1 mg/mL in 1M guanidine.HCl, 0.1M TRIS, pH=8.6 at roomtemperature in the presence of air. After stirring overnight, folding iscomplete. Alternatively, full length peptide preferably is folded in 2Mguanidine.HCl, 0.1 M TRIS, pH 8 containing 8 mM cysteine and 1 mMcystine at 0.5 mg/ml at room temperature with stirring overnight.

[0094] Validation procedures used to confirm purity and chemicalstructure include HPLC, electrospray mass spectrometry, and peptidemapping. Biological activity of the cross-over chemokines isdemonstrated following standard chemotaxis and receptor binding assaysusing recombinant or chemically synthesized MPBV, SDF-1α and/or RANTESas controls.

Example 3

[0095] Modular Synthesis of Cross-over Chemokines Comprising FunctionalModules from vMIP-I And vMIP-II

[0096] Novel viral cross-over chemokines are constructed by combiningsegments comprising functional modules from two related virally encodedchemokines. Functional protein modules corresponding to homologousbinding sites on the surface of native chemokines are identified byalignment of segments (halves) of Macrophage Derived Chemokine (MDC) andthe Kaposi's sarcoma-associated herpes virus (KSHV) chemokines againstother known chemokines. Sequence alignment of the viral chemokinesagainst the RANTES three-dimensional structure (Brookhaven ProteinDatabank, Brookhaven National Labs, NY) using LOOK(® software identifiedsections of sequences that correlated with patches (putative bindingsites) localized to the surface of the folded chemokines. Crossoverchemokines are made by modular synthesis using native ligation at thecentral cysteine and folding of viral chemokine segments derived fromvMIP-1 (MPAV) and vMIP-II (MPBV). The two unique crossover chemokinesare designated MP(A/B)V and MP(B/A)V. The MP(A/B)V cross-over chemokinecomprises the N-terminal segment from MPAV (amino acids 1-35) and theC-terminal segment of the MPBV (amino acids 38-74). The MP(B/A)Vcross-over chemokine comprises the N-terminal segment from MPBV (aminoacids 1-37) and the C-terminal segment of the MPAV (amino acids 36-71).The effect of these crossovers on the three-dimensional (tertiary)structure of a chemokine are evaluated relative to the three-dimensionalscaffold, which represented separation of functional modulescorresponding to the “N-terminal tail” and the “lower right side” in theN-terminal segment from the “front upper left” in the C-terminalsegment. The amino acid sequences for four chemically synthesizedchemokines are shown in Table II below and represent two of the nativevirally encoded chemokines MPAV and MPBV, and two of the cross-overchemokines corresponding to MP(A/B)V and MP (B/A)V. TABLE II Amino acidsequences of the native MPAV and MPBV molecules, and cross-overchemokines MP(A/B)V and MP(B/A)V. MPAV (1-71) (SEQ ID NO:1):AGSLVSYTPNSCCYGFQQHPPPVQILKEWYPTSPA C PKPGVILLTKRGRQICADPSKNWVRQLMQRLPAIA MPBV (1-74) (SEQ ID NO:2):GDTLGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLCSKPGVIFLTKRGRQVCADKSKDWVKKLMQQLPVTAR MP (A/B) V(1-72) (SEQ ID NO:3):AGSLVSYTPNSCCYGFQQHPPPVQILKEWYPTSPA CSKPGVIFLTKRGRQVCADKSKDWVKKLMQQLPVTAR MP (B/A) V(1-73) (SEQ ID NO:4):GDTLGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLC PKPGVILLTKRGRQICADPSKNWVRQLMQRLPAIA

Example 4

[0097] Modular Synthesis of Cross-over Chemokines Comprising FunctionalModules from SDF-1α and RANTES

[0098] All CC and CXC chemokines contain four cysteines giving sitesamenable to native chemical ligation at Xxx-Cys positions. Peptidescorresponding to the N-terminal and C-terminal halves flanking the Cyspositions are synthesized, purified and ligated following the schemedepicted in Tables III-V and FIGS. 1 and 2. In particular, SDF-1α (a CXCchemokine that binds to the CXCR4 receptor) and RANTES (a CC chemokinethat binds to the CCR5 receptor) are employed in the modular synthesisof cross-over chemokines using eight N-terminal modules in variouscombinations with four C-terminal modules derived from these chemokines(see Tables IV and V). Additional diversity is incorporated into theN-terminal segment by the deletion of the “X” residue from the “CXC”module of SDF-1α and insertion of a residue between the “CC” module ofRANTES, for a total of eight N-terminal modules. For example, in SDF-1αthe N-terminal module corresponds to “KPVSLSYRCP” from which the Presidue is deleted to give KPVSLSYRC (i.e., deletion of the “X” residuefrom the “CXC” module); in RANTES the N-terminal module corresponds to“SPYSSDTTPC” into which a P residue is inserted to yield “SPYSSDTTPCP”(i.e., insertion of an “X” residue between the “CC” module). Nativechemical ligation technology is used to synthesize the two modifiednative and 30 hybrid chemokines between SDF-1α and RANTES. In addition,solid phase chemical ligation is used to construct the two modifiednative molecules for comparison to molecules prepared by native chemicalligation. The cross-over chemokines synthesized are assayed for bindingto the CXCR4 and CCR5 receptors, and the residues directly involved inbinding to the two different receptors are identified (see Example 5).This library of molecules also is used to probe the structure andfunction of the N-terminal CXC or CC modules, the hydrophobic pocket,and the C-terminal regions between the two classes of chemokines. Inaddition, the hybrid chemokines are screened to identify those moleculeswhich display “dual functionality,” i.e., the ability to bind both CXCR4and CCR5. Selection of the hybrid chemokines is characterized usingIH-NMR and other biophysical techniques. This first group of moleculesare used in a second round of iteration (for example N-terminalmodifications) to further improve binding to the receptors. Use of thecross-over chemokine molecules also are assayed for blocking of CXCR4and CCR5 for prevention of HIV entry into cells, as binding ofchemokines to CXCR4 and CCR5 has been shown to block HIV entry intocells (Simons et al., Science (1997) 275:1261-1264) (see Example 5).Other biological assays may be used to determine generalstructure-function relationships within chemokine molecules. TABLE IIIAmino acid sequences for native and base synthetic SDF-1 and RANTESSDF-1α (human residues 1-93): MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVAPA(SEQ ID NO:5) NVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEK ALNKRFKMSDF-1α (1-67) (synthetic base molecule missing pre-sequence/N-terminalresidues 1-21 and C-terminal residues 89-93): (SEQ ID NO:6)KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKN NNRQVCIDPKILKWIQEYLEKALNRANTES (human residues 1-91): (SEQ ID NO:7)MKVSAARLAVILIATALCAPASASPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSL EMS RANTES (1-68) (syntheticbase molecule missing pre-sequence/N-terminal residues 1-23):SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK (SEQ ID NO:8)NRQVCANPEKKWVREYINSLEMS

[0099] TABLE IV Modular synthesis of cross-over chemokines using eightN-terminal modules in combination with four C-terminal modules toconstruct cross-over chemokine molecules. Ref Amino Acid Sequence SEQ IDNO 8X N-terminal modules: SS KPVSLSYRCPCRFFESHVARANVKHLKILNTPN (SEQ IDNO: 9) S'S KPVSLSYRCCRFFESHVARANVKHLKILNTPN (SEQ ID NO: 10) SRKPVSLSYRCPCFAYIARPLPRAHIKEYFYTSGK (SEQ ID NO: 11) S'RKPVSLSYRCCFAYIARPLPRAHIKEYFYTSGK (SEQ ID NO: 12) RRSPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGK (SEQ ID NO: 13) R'RSPYSSDTTPCPCFAYIARPLPRAHIKEYFYTSGK (SEQ ID NO: 14) RSSPYSSDTTPCCRFFESHVARANVKHLKILNTPN (SEQ ID NO: 15) R'SSPYSSDTTPCPCRFFESHVARANVKHLKILNTPN (SEQ ID NO: 16) 4X C-terminalmodules: SS CALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN (SEQ ID NO: 17) SRCALQIVARLKNNNRQVCANPEKKWVREYINSLEMS (SEQ ID NO: 18) RSCSNPAVVFVTRXNRQVCIDPKLKWIQEYLEKALN (SEQ ID NO: 19) RRCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS (SEQ ID NO: 20)

[0100] TABLE V Amino acid sequences for SDF-1α/RANTES cross-overmolecules Combination of 8X N-terminal and 4X C-terminal modules: SSSS(control) SRSS RRSS RSSS SSSR SRSR RRSR RSSR SSRS SRRS RRRS RSRS SSRRSRRR RRRR (control) RSRR S'SSS S'RSS R'RSS R'SSS (−Pro control) S'SSRS'RSR R'RSR R'SSR S'SRS S'RRS R'RRS R'SRS S'SRR S'RRR R'RRR R'SRR (+Procontrol) SSSS: KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQLVARLKN (SEQ ID NO:21) NNRQVCIDPKLKWIQEYLEKALN SSSR:KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKN (SEQ ID NO: 22)NNRQVCANPEKKWVREYINSLEMS SSRS:KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCSNPAVVFVTR (SEQ ID NO: 23)KNRQVCIDPKLKWIQEYLEKALN SSRR:KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCSNPAVVFVTR (SEQ ID NO: 24)KNRQVCANPEKKWVREYINSLEMS S'SSS:KPVSLSYRCCRFFESHVARANVKHLKILNTPNCALQIVARLKN (SEQ ID NO: 25)NNRQVCIDPKLKWIQEYLEKALN SSSR:KPVSLSYRCCRFPESHVARANVKHLKILNTPNCALQIVARLKN (SEQ ID NO: 26)NNRQVCANPEKKWVREYINSLEMS SSRS:KPVSLSYRCCRFFESHVARANVKHLKILNTPCNSNPAVVFVTR (SEQ ID NO: 27)KNRQVCIDPKLKWIQEYLEKALN S'SRR:KPVSLSYRCCRFFESHVARANVKHLKILNTPNCSNPAVVFVTR (SEQ ID NO: 28)KNRQVCANPEKKWVREYINSLEMS SRSS:KPVSLSYRCPCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKN (SEQ ID NO: 29)NNRQVCIDPKLKWIQEYLEKALN SRSR:KPVSLSYRCPCFAYIARPLPRAHLKEYFYTSGKCALQIVARLKN (SEQ ID NO: 30)NNRQVCANPEKKWVREYINSLEMS SRRS:KPVSLSYRCPCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTR (SEQ ID NO: 31)KNRQVCIDPKLKWIQEYLEKALN SRRR:KPVSLSYRCPCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTR (SEQ ID NO: 32)KNRQVCANPEKKWVREYINSLEMS S'RSS:KPVSLSYRCCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKNN (SEQ ID NO: 33)NRQVCIDPKLKWIQEYLEKALN S'RSR:KPVSLSYRCCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKNN (SEQ ID NO: 34)NRQVCANPEKIKWVREYINSLEMS S'RRS:KPVSLSYRCCFAYIARPLPRAFIIKEYFYTSGKCSNPAVVFVTRK (SEQ ID NO: 35)NRQVCIDPKLKWIQEYLEKALN S'RRR:KPVSLSYRCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK (SEQ ID NO: 36)NRQVCANPEKKWVREYTNSLEMS RRSS:SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKNN (SEQ ID NO: 37)NRQVCIDPKLKWIQEYLEKALN RRSR:SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKNN (SEQ ID NO: 38)NRQVCANPEKKWVREYTNSLEMS RRRS:SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK (SEQ ID NO: 39)NRQVCIDPKLKWIQEYLEKALN RRRR:SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK (SEQ ID NO: 40)NRQVCANPEKXWVREYINSLEMS R'RSS:SPYSSDTTPCPCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKN (SEQ ID NO: 41)NNRQVCIDPKLKWIQEYLEKALN R'RSR:SPYSSDTTPCPCFAYIARPLPRAHIKEYFYTSGKCALQIVARLKN (SEQ ID NO: 42)NNRQVCANPEKKWVREYINSLEMS R'RRS:SPYSSDTTPCPCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTR (SEQ ID NO: 43)KINRQVCIDPKLKWIQEYLEKALN R'RRR:SPYSSDTTPCPCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTR (SEQ ID NO: 44)KNRQVCANPEKKWVREYINSLEMS RSSS:SPYSSDTTPCCRFFESHVARANVKHLKILNTPNCALQIVARLICN (SEQ ID NO: 45)NNRQVCLDPKLKWIQEYLEKALN RSSR:SPYSSDTTPCCRFFESHVARANVKHLKILNTPNCALQIVARLKN (SEQ ID NO: 46)NNRQVCANPEKKWVREYINSLEMS RSRS:SPYSSDTTPCCRFFESHVARANVKHLKILNTPNCSNPAVVFVTR (SEQ ID NO: 47)KNRQVCIDPKLKWIQEYLEKALN RSRR:SPYSSDTTPCCREFESHVARANVKHLKILNTPNCSNPAVVFVTR (SEQ ID NO: 48)KNRQVCANPEKKWVRLEYINSLEMS R'SSS:SPYSSDTTPCPCRFFESHVARANVKHLKILNTPNCALQIVARLK (SEQ ID NO: 49)NNNRQVCIDPKLKWIQEYLEKALN R'SSR:SPYSSDTTPCPCRFFESHVARANVKHLKILNTPNCALQIVARLK (SEQ ID NO: 50)NNNRQVCANPEKKWVREYINSLEMS R'SRS:SPYSSDTTPCPCRFFESHVARANVKHLKILNTPNCSNPAVVFVT (SEQ ID NO: 51)RKNRQVCIDPKLKWIQEYLEKALN R'SRR:SPYSSDTTPCPCRFFESHVARANVKHLKILNTPNCSNPAVVFVT (SEQ ID NO: 52)RKNRQVCANPEKKWVREYINSLEMS

[0101] Ligation and proper folding of a small library of cross-overchemokines are demonstrated in FIGS. 4-5, which show analytical HPLC forthe SSSS (control), S′SSS (-Pro control), SRRR, S′RRR, RRRR (control),R′RRR (-Pro control), RSSS, and R′SSS chemokines depicted in Table V.Analytical HPLC also demonstrates variable separation properties amongthe cross-over chemokines, reflecting a likely difference in in vivofunctionality. The calculated molecular weight (MW) of the expectedcross-over protein ligation products and the actual MW determined byelectrospray mass spectroscopy show a high level of agreement (See,e.g., Table VI). TABLE VI Calculated and Measured Molecular Weights forModular Cross-Over Chemokines Modular Calculated Measured Chemokine MW(Dalton) MW (Dalton) SSSS (control) 7788.28 7789.29 S'SSS (−Pro control)7691.16 7692.63 SRRR 7939.34 7939.96 S'RRR 7842.22 7842.09 RRRR(control) 7847.06 7848.36 R'RRR (+Pro control) 7944.17 7945.63 RSSS7696.00 7695.06 R'SSS 7793.12 7791.96

Example 5

[0102] Cross-Over Chemokine Assays

[0103] Chemotaxis Assays:

[0104] Human peripheral blood leukocytes are isolated from normal donorsaccording to established protocols for purification of monocytes, Tlymphocytes and neutrophils. A panel of CC and CXC chemokinereceptor-expressing test cells is constructed and evaluated followingexposure to serial dilutions of individual compounds from the library ofcross-over chemokines RANTES/SDF-1α, MP(A/B)V and MP (B/A)V. Syntheticnative RANTES, SDF-1α, MPAV and MPBV are used as controls. The panel ofcells represent human kidney embryonic epithelial (HEK) 293 cellstransfected with expression cassettes encoding various chemokinereceptors including CXCR4/Fusion/LESTR, CCR3, CCR5, CXC4 (these cellsare available from various commercial and/or academic sources or can beprepared following standard protocols). Leukocyte migration relative tothe transfected HEK cells is evaluated using a 48-well microchamber;migration of the receptor transfected HEK 293 cells also is assessed bythe 48-well microchamber technique with the polycarbonate filters (10 umpore-size) precoated with Collagen type I (Collaborative BiomedicalProducts, Bedford, Mass.)(Neote et al., Cell (1993) 72:415-425; Risau etal., Nature (1997) 387:671-674; Angiololo et al., Annals NY Acad Sci.(1996) 795:158-167; Friedlander et al., Science (1995) 870:1500-1502).The results are expressed as the chemotaxis index (CI) representing thefold increase in the cell migration induced by stimuli versus controlmedium. All experiments are performed at least two times and resultsfrom one experiment are shown. The statistical significance of thedifference between migration in response to stimuli and control areaccessed by Student's T test.

[0105] Receptor Binding Assays:

[0106] Receptor binding assays are performed using a singleconcentration of ¹²⁵I labeled chemokines in the presence of increasingconcentrations of unlabeled ligands following standard protocols. Thebinding data are analyzed, for example, with a computer program such asLIGAND (P. Munson, Division of Computer Research and Technology, NIH,Bethesda, Md.). The binding data are subjected to Scatchard plotsanalysis with both “one site” and “two site” models compared to nativeleukocytes or the panel of receptor-transfected HEK cells expressingCXCR4, CCR3, CCR5 or CXC4. The rate of competition for binding byunlabeled ligands is calculated with the following formula: %inhibition=1-(Binding in the presence of unlabeled chemokine/binding inthe presence of medium alone)×100.

[0107] HIV-1 Inhibition Assays:

[0108] Chemokine receptors act as co-receptors for human immunedeficiency virus type (HIV)-1 entry into CD4+cells. The CC chemokinesMIP-1A, MIP-1B, RANTES and eotaxin can suppress infection by somestrains of HIV in PBMCs and chemokine receptor transfected cell lines.The viral-produced chemokine vMIP-1 inhibits some primary non-syncytiuminducing (NSI) HIV strains when co-transfected with the NSI strain HIV-1co-receptor CCR5. CCR3 is the predominant chemokine receptor throughwhich eotaxin, RANTES and other CC chemokines activate eosinophils.RANTES and MIP-1A also can utilize the CCR1 receptor that is expressedon eosinophils. In addition, synthetic N-terminal variants of CC (e.g.Met-RANTES) and CXC (e.g. IL-8) chemokines function as receptorantagonists on eosinophils and neutrophils, whereas the nativestructures do not. Similarly, the CXC chemokine SDF-1α is a potentchemoattractant for leukocytes through activation of the receptorCXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of HIV-1.CXCR4 mediated HIV-1 fusion can be inhibited in some cells by SDF-1α.Thus, despite the sequence similarities between certain chemokines ofthe same family, the binding and antagonist/agonist properties for HIVinfection vary significantly.

[0109] Compounds from the library of cross-over chemokinesRANTES/SDF-1α, MP(A/B)V and MP (B/A)V are screened for receptor usage,inhibition of HIV infection, potency and breadth of activity against HIVinfection, induction of calcium mobilization and angiogenesis. Theassays are used to evaluate suppression of HIV-I infection/replicationin U87/CD4 cells (a human glioma cell line) expressing HIV-1co-receptors and also in primary peripheral blood mononuclear cells(PBMCs).

[0110] The receptor-transfected U87/CD4 cells are obtainable bytransfecting cells with an expression cassette encoding the respectivereceptors following standard protocols. The cells are maintained inDulbecco's Minimal Essential Medium containing 10% FCS, glutamine,antibiotics, 1 ug/ml puromycin (Sigma Chemicals) and 300 ug/ml neomycin(G418; Sigma) and split twice a week. PBMCs are isolated from healthyblood donors by Ficoll-Hypaque centrifugation, then stimulated for 2-3days with phytohemagglutinin (PHA) (5ug/ml) and IL-2 (100 U/ml)(Simmons,et al, J. Viorol (1996) 70:8355-8360). CD4+ T-cells are purified fromthe activated PBMC by positive selection using anti-CD4 immunomagneticbeads (DYNAL Inc.), screened for CCR-5 defective alleles, and cells fromallele defective or wild-type donors used depending on the assay. HIVisolates are obtainable from various sources including the NIAID HIV-1Antigenic Variation study, or from similar programs organized by the USDepartment of Defense or the World Health Organization. Phenotypes oftest viruses are tested by their ability to form syncytia (SI) in MT-2cells that are cultured in RPMI 1640 medium containing 10% fetal calfserum (FCS), glutamine and antibiotics. and split twice a week.Recombinant human CC-chemokines MIP-1A, MIP-1B and RANTES are obtainablefrom R&D Systems Inc. (Minneapolis). Synthetic SDF-1α stocks areobtainable from Gryphon Sciences (M.A.S. and D.A.T.) and BerlexBiosciences (R.H.). Chemokine stocks are compared for purity andpotency.

[0111] Assay for inhibition of HIV infection:

[0112] Compounds from the library of cross-over chemokinesRANTES/SDF-1α, MP(A/B)V and MP (B/A)V are tested against a panel ofU87/CD4 cells stably expressing either CCR3, CCR5, CXC4 or CXCR4receptors exposed to HIV-1/NSI strains SL-2 and SF162 (macrophage-tropic strains that utilize the RANTES, MIP-1α and MIP-1β receptor CCR5to gain entry into CD4+cells) and the dual-tropic syncytium inducing(SI) strains 89.6 and 2028 (SI dual tropic strains that can use CXCR4and CCR3 in addition to CCR5 for entry). Lymphocytes and CD4+ T-cellsfrom donors also are tested. Serial concentrations ranging from 0 to 500nM of the cross-over proteins are used. RANTES, MPBA, MPBV and SDF-1αare used as controls. Inhibition of HIV infection is reported as apercentage of infection relative to modular protein and controlconcentrations.

[0113] Purified lymphocytes are stimulated with PHA (0.5ug/ml) andcultured for 2-3 days at 2×10⁶/ml in medium containing IL-2(Boeringer-Mannheim, 20 U/ml) before being used in infection assays.Cells are pre-treated with appropriate concentrations of chemokines for30 minutes at 37° C. Approximately 400-1000 TCID of virus are added toan appropriate volume and incubated at 37° C. for 3 hours. Cells arethen washed 4 times and resuspended in an appropriate volume of mediacontaining IL-2 and relevant chemokine at the appropriate concentration.Cells are fed every 3 days with fresh medium contain IL-2 and chemokine.From days 3 through 7 post-infection, the cultures are examinedmicroscopically for syncytium formation and the supernatant analyzed forp24 antigen production using an enzyme linked immunoabsorbent assay(ELISA)(McKnight et al., Virology (1994) 201:8-18). Inhibitory doses acalculated relative to the final concentration of chemokine in theculture on day 0. Virus production in the absence of chemokine isdesignated as 100%, and the ratios of p24 antigen production inchemokine-containing cultures calculated relative to this percentage.The chemokine concentrations (pg/ml) causing 50% and 90% reduction inp24 antigen production are determined by linear regression analysis. Ifthe appropriate degree of inhibition is not achieved at the highest orlowest chemokine concentration, a value of>or<is recorded.

[0114] Virus infectivity on the receptor expressing U87/CD4 cells isassessed by focus-forming units (FFU) (Simmons, et al., Science (1997)276:276-279). The FFU for viruses using more than one co-receptor isassessed separately for each appropriate co-receptor expressing U87/CD4cell type. Cells are seeded into 48 well trays at 1×10⁴ cells/wellovernight. The cells are then pre- treated for 30 minutes at 37° C. withappropriate concentrations of chemokine in 75ul. 100 FFU of each virusin 75ul is added and incubated for 3 hours at 37° C. Cells are washed 3times and 500ul of medium containing the appropriate chemokine at thecorrect concentration is added. After 5 days the cells are fixed for 10minutes in cold acetone:methanol (1:1) and analyzed for p24 antigenproduction. Standard errors are estimated from duplicate wells andresults presented are representative of three separate experiments.

[0115] Assay for breadth and potency of cross-over chemokines againstHIV infection:

[0116] The breadth and potency of the inhibitory actions of compoundsfrom the library of cross-over chemokines RANTES/SDF-1α, MP(A/B)V and MP(B/A)V are tested against native CC-chemokines (MIP-1A, MIP-1B andRANTES) for M-tropic primary isolates of HIV-1, and against a nativeCXC-chemokine (SDF-1α) for T-tropic isolates in mitogen-stimulatedprimary CD4+ T-cells. The cross-over chemokines are evaluated for theirpotency and spectrum of agonistic activity against HIV-1 strainsrelative to the native CC- and CXC- chemokines to identify the mostactive inhibitor of HIV-1 replication and the best template fortherapeutic development. The properties and activities of M-Tropic andT-tropic primary HIV-1 isolates are recorded and compared to inhibitionof infection by exposure to the cross-over chemokines relative to theHIV isolate designation, genetic subtype, and phenotype determined byability of an isolate to form (SI) or not form (NSI) syncytia in MT-2cells, the ability of an isolate to replicate efficiently in activatedCD4+ T-cells from individuals homozygous for either wild-type ordelta-32 CCR5 alleles, and the ability of an isolate to replicate inU87/CD4 cells stably expressing either CCR5 or CXCR4. The median ID50and ID90 values (ng/ml) are calculated for each sample. A valueof>indicates that 50% or 90% inhibition is not achieved at a chemokineconcentration of the highest tested in any experiment. A valueof<indicates that 50% or 90% inhibition is always achieved at achemokine concentration of the lowest tested. The genetic subtypes ofthe test isolates and their abilities to use CXCR4 and CCR5 to entertransfected U87MG-CD4 cells are also compared. The means from twoindependent experiments are compared. FACS analysis of CCR5 and CXCR4receptor expression levels, and/or competitive inhibition assay ofcross-over chemokines and receptor down-regulation also may be testedfollowing standard protocols (Wu et al., J. Exp. Med. (1997)185:168-169; and Trkola et al., Nature (1996) 384:184-186).

[0117] Assay for Measuring Changes in Intracellular CalciumConcentration ([Ca2+]):

[0118] Calcium mobilization is indicative of receptor binding. Compoundsfrom the library of cross-over chemokines RANTES/SDF-1α, MP(A/B)V and MP(B/A)V are assayed for calcium mobilization in purified neutrophils andeosinophils following standard protocols (Jose et al., J Exp Med (1994)179:881-887). Purified neutrophils or eosinophils are incubated withfura-2 acetoxymethyl ester (1-2.5uM), washed 3 times in 10 mM PBS(without Ca2+/Mg2+)+0.1% BSA (200 ×g, 8 min), and finally resuspended at2×10⁶ cells/ml in 10 mM PBS (without Ca2+/Mg2+)+0.25% BSA+10 mM HEPES+10mM glucose. Aliquots of cells are placed in quartz cuvettes and theexternal Ca2+ concentration adjusted to 1 mM with CaCl₂ Changes influorescence are measured at 37° C. using a fluorescencespectrophotometer at excitation wavelengths 340 nm and 380 nm andemission wavelength 510 nm. [Ca2+] levels are calculated using the ratioof the two fluorescence readings and a K for Ca2+ at 37° C. of 224 nM.

[0119] CAM assay for angiogenic activity:

[0120] Angiogenic activities of compounds from the library of cross-overchemokines RANTES/SDF-1α, MP(A/B)V and MP (B/A)V are evaluated by thechick chorioallantoic membrane (CAM) assay (Oikawa et al., Cancer Lett(1991) 59:57-66). Native chemokines are used as controls. FertilizedPlymouth Rock x while Leghorn eggs are incubated at 37° C. in ahumidified atmosphere (relative humidity, approx. 70%). Test samples aredissolved in sterile distilled water or PBS. Sterilized sample solutionis mixed with an equal volume of autoclaved 2% methylcellulose.Additional controls are prepared with vehicle only (1% methylcellulosesolution). 20ul of the sample solution is dropped on parafilm and driedup. The methylcellulose disks are stripped off from the parafilm andplaced on a CAM of a 10 or 11 day old chick embryo. After 3 days, theCAMs are observed by means of an Olympus stereoscope. A 20% fat emulsion(Intralipos 20%, Midori-Juji, Osaka, Japan) is injected into the CAM toincrease the contrast between blood and surrounding tissues (Danesi etal., Clin Cancer Research (1997) 3:265-272). The CAMs are photographedfor evaluation of angiogenic response. Angiogenic responses are gradedas negative, positive or unclear on the basis of infiltration of bloodvessels into the area of the implanted methylcellulose disk by differentobservers.

[0121] As exemplified above, modular protein libraries comprisingcross-over molecules are constructed. The cross-over libraries find usein identifying novel proteins having cross-over activities contributedby a combination of individual functional protein modules from two ormore distinct proteins of the same family or class. The methods of theinvention can be readily adapted and integrated with genomic sequencingand bioinformatics to prepare novel combinatorial modular proteinlibraries for identifying new drug candidates, and for evaluating andvalidating the physiological relevance of the new targets. This approachrepresents an advance over traditional discovery protocols that rely onnative, historical, and/or random synthetic libraries subjected to massscreening. Generation of modular protein libraries representing afocused set of molecules decreases the time and cost of discoveringnovel therapeutic agents for multiple disease states. The modularsynthesis approach and the construction of cross-over protein librariesgreatly expands the range of compounds available for biologicalscreening and discovery of pharmaceutical agents.

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[0213] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asof each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0214] The invention now having been fully described, it will beapparent to one or ordinary skill in the art that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A cross-over protein produced by chemicalligation of two or more functional protein modules derived from two ormore different parent protein molecules.
 2. The cross-over protein ofclaim 1, wherein said parent protein molecules are of the same family ofprotein molecules.
 3. The cross-over protein of claim 2, wherein saidchemical ligation is selected from the group consisting of nativechemical ligation, oxime forming chemical ligation, thioester formingligation, thioether forming ligation, hydrazone forming ligation,thaizolidine forming ligation, and oxazolidine forming ligation.
 4. Thecross-over protein of claim 1, wherein said cross-over protein comprisesa chemical tag.
 5. The cross-over protein of claim 4, wherein saidchemical tag is a detectable label.
 6. The cross-over protein of claim5, wherein said detectable label comprises an unnatural amino acid. 7.The cross-over protein of claim 6, wherein said unnatural amino acidcomprises a chromophore.
 8. The cross-over protein of claim 7, whereinsaid chromophore is an acceptor moiety of an acceptor-donor resonanceenergy transfer pair.
 9. The cross-over protein of claim 7, wherein saidchromophore is a donor moiety of an acceptor-donor resonance energytransfer pair.
 10. The cross-over protein of claim 4, wherein saidchemical tag comprises a chemical handle for attaching said cross-overprotein to a support matrix.
 11. The cross-over protein of claim 10,wherein said cross-over protein is attached to a support matrix via saidchemical handle.
 12. The cross-over protein of claim 11, wherein saidcross-over protein is attached to a support matrix via said chemicalhandle in a spatially addressable array.
 13. The cross-over protein ofclaim 1, wherein the protein is a cross-over chemokine.
 14. Thecross-over protein of claim 13, wherein said cross-over chemokinecomprises a functional protein module of a chemokine selected from thegroup consisting of RANTES, SDF1, and MIP.
 15. The cross-over protein ofclaim 14, wherein said functional protein module comprises an N-terminalmodule corresponding to a SEQ ID NO selected from the group consistingof SEQ ID NO: 9-16.
 16. The cross-over protein of claim 14, wherein saidfunctional protein module comprises an C-terminal module correspondingto a SEQ ID NO selected from the group consisting of SEQ ID NO: 17-20.17. The cross-over protein of claim 14, wherein said cross-overchemokine corresponds to a SEQ ID NO selected from the group consistingof SEQ ID NO: 3, 4, 22-24, 26-39, 41-43 and 45-52.
 18. A protein librarycomprising a collection of cross-over proteins of claim
 1. 19. Theprotein library of claim 18, wherein said collection of cross-overproteins comprises two or more unique cross-over proteins.
 20. Theprotein library of claim 19, wherein one or more of said uniquecross-over proteins is produced by chemical ligation of two or moreN-terminal peptide segments comprising one or more functional proteinmodules of a first parent protein and two or more C-terminal peptidesegments comprising one or more functional protein modules of a secondparent protein.
 21. The protein library of claim 18, wherein thecross-over proteins comprise cross-over chemokines.
 22. The proteinlibrary of claim 21, wherein said cross-over chemokines comprise afunctional protein module of a chemokine selected from the groupconsisting of RANTES, SDF1 and MIP.
 23. The protein library of claim 22,wherein said functional protein module comprises an N-terminal modulecorresponding to a SEQ ID NO selected from the group consisting of SEQID NO: 9-16.
 24. The protein library of claim 22, wherein saidfunctional protein module comprises an C-terminal module correspondingto a SEQ ID NO selected from the group consisting of SEQ ID NO: 17-20.25. The protein library of claim 22, wherein one or more of saidcross-over chemokines correspond to a SEQ ID NO selected from the groupconsisting of SEQ ID NO: 3, 4, 22-24, 26-39, 41-43 and 45-52
 26. Apharmaceutical composition comprising a cross-over protein according toany one of claims 13-17.
 27. A kit comprising a cross-over proteinaccording to any one of claims 1-26.
 28. A method of producing across-over protein, said method comprising: ligating underchemoselective chemical ligation conditions (i) at least one N-terminalpeptide segment comprising a functional protein module derived from afirst parent protein, and (ii) at least one C-terminal peptide segmentcomprising a functional protein module derived from a second parentprotein having an amino acid sequence that is different from said firstparent protein, wherein said N-terminal peptide segment and saidC-terminal peptide segment comprise compatible reactive groups capableof chemoselective chemical ligation, whereby a covalent bond is formedbetween said N-terminal peptide segment and said C-terminal peptidesegment so as to produce a chemical ligation product comprising across-over protein.
 29. The method of claim 28 further comprising thestep of repeating said ligating one or more times with one or moresecond peptide segments selected from the group consisting of anN-terminal peptide segment and a C-terminal peptide segment.
 30. Themethod of claim 28, wherein said parent protein molecules are of thesame family of protein molecules.
 31. The method of claim 28, whereinsaid chemoselective chemical ligation is selected from the groupconsisting of native chemical ligation, oxime forming chemical ligation,thioester forming ligation, thioether forming ligation, hydrazoneforming ligation, thaizolidine forming ligation, and oxazolidine formingligation.
 32. A method of producing a cross-over protein library, saidmethod comprising: ligating under chemoselective reaction conditions aplurality of unique N-terminal peptide segments comprising one or morefunctional protein modules derived from first parent protein and aplurality of unique C-terminal peptide segments comprising one or morefunctional protein modules derived from a second parent protein havingan amino acid sequence that is different from said first parent protein,wherein said N-terminal peptide segments and said C-terminal peptidesegments comprise compatible reactive groups capable of chemoselectivechemical ligation, whereby a covalent bond is formed between saidN-terminal peptide segments and said C-terminal peptide segments so asto produce a plurality of chemical ligation products comprising aplurality of unique cross-over proteins.
 33. The method of claim 32,wherein said plurality of N-terminal peptide segments are obtained bycross-over ligation of two or more different parent protein molecules.34. The method of claim 32, wherein said plurality of C-terminal peptidesegments are obtained by cross-over ligation of two or more differentparent protein molecules.
 35. The method of claim 32, wherein saidparent protein molecules are of the same family of protein molecules.36. The method of claim 32, wherein said chemoselective chemicalligation is selected from the group consisting of native chemicalligation, oxime forming chemical ligation, thioester forming ligation,thioether forming ligation, hydrazone forming ligation, thaizolidineforming ligation, and oxazolidine forming ligation.
 37. A method ofscreening a cross-over protein library, said method comprising:contacting a receptor with one or more cross-over proteins obtained froma cross-over protein library, and identifying a cross-over protein fromsaid library that is a ligand for said receptor in an assaycharacterized by detection of binding of said ligand to said receptor.38. The method of claim 37, wherein one or more of said cross-overproteins comprise a detectable label.
 39. The method of claim 38,wherein said detectable label comprises a chromophore.
 40. The method ofclaim 38, wherein said detectable label comprises an unnatural aminoacid.
 41. The method of claim 40, wherein said unnatural amino acidcomprises a chromophore.
 42. The method of claim 39, wherein saidchromophore is an acceptor moiety of an acceptor-donor resonance energytransfer pair.
 43. The method of claim 41, wherein said chromophore is adonor moiety of an acceptor-donor resonance energy transfer pair. 44.The method of claim 39, wherein said detection is fluorescencedetection.
 45. The method of claim 44, wherein said fluorescencedetection is fluorescence resonance energy transfer detection.
 46. Themethod of claim 37, wherein said screening is high throughput.
 47. Themethod of claim 37, wherein said cross-over protein library comprisesone or more cross-over chemokines.
 48. The method of claim 47, whereinsaid cross-over chemokines comprise a functional protein module of achemokine selected from the group consisting of RANTES, SDF1, and MIP.49. The method of claim 48, wherein said functional protein modulecomprises an N-terminal module corresponding to a SEQ ID NO selectedfrom the group consisting of SEQ ID NO: 9-16.
 50. The method of claim48, wherein said functional protein module comprises an C-terminalmodule corresponding to a SEQ ID NO selected from the group consistingof SEQ ID NO: 17-20.
 51. The method of claim 48, wherein said cross-overchemokine corresponds to a SEQ ID NO selected from the group consistingof SEQ ID NO: 3, 4, 22-24, 26-39, 41-43 and 45-52.